Endotrophin neutralization and use thereof

ABSTRACT

Aspects of the present invention relate to methods and reagents for increasing chemosensitivity to platinum-based chemotherapy. In one aspect, a method of increasing chemosensitivity to platinum-based chemotherapy is provided, comprising administering to a patient in need thereof an effective amount of an endotrophin-neutralizing agent. The agent can be a monoclonal antibody, or fragment thereof, capable of binding to the C5 domain of the alpha3 chain of collagen VI. In some embodiments, the method can further include administering an effective amount of thiazolidinedione to said patient.

This application is a continuation of U.S. application Ser. No.14/306,784, filed Jun. 17, 2014, which claims benefit of priority toU.S. Provisional Application Ser. No. 61/956,807, filed Jun. 17, 2013.The entire contents of the applications references above are herebyincorporated by reference.

STATEMENT OF GOVERNMENT LICENSE RIGHTS

This invention was made with government support under Grant No. USAMRMCBC085909 awarded by the Department of Defense. The government hascertain rights in the invention.

INCORPORATION OF SEQUENCE LISTING

The sequence listing that is contained in the file named“UTSH0303USC1_ST25.txt”, which is 13 KB (as measured in MicrosoftWindows®) and was created on Jan. 27, 2017, is filed herewith byelectronic submission and is incorporated by reference herein.

BACKGROUND

The platinum-based chemotherapeutic agent cisplatin(cis-diammine-dichloro-platinum) has been well established in clinicaltreatment regimens due to its effectiveness on human tumor cells, suchas in the context of ovarian, lung, testicular and breast cancer(Kelland, 2007; Lee et al, 2004; Sirohi et al, 2008). Cisplatin triggersformation of intra-strand and inter-strand DNA-adducts, which leads tocell cycle arrest, followed by apoptosis (Kelland, 2007). However, aninherent or acquired resistance to cisplatin is a major clinicaldrawback for patients who relapse after an initial favorable responses(Galluzzi et al, 2012). Cisplatin resistance is a complex problem whichinvolves multiple pathways including increased drug efflux, evasion ofapoptotic pathways, a bypass of the replication checkpoint, increasedcell proliferation and increased DNA damage repair (Galluzzi et al,2012). To overcome the drug resistance against platinum-basedchemotherapy, combination therapies with peroxisomeproliferator-activated receptor gamma (PPARγ) agonists, thethiazolidinediones (TZDs), have been performed. The basis for thisapproach is the growth inhibitory effect of these PPARγ agonists ontransformed cells through both PPARγ-dependent and -independent pathways(Blanquicett et al, 2008; Mueller et al, 1998; Palakurthi et al, 2001;Satoh et al, 2002). PPARγ is a member of the nuclear hormone receptorsuperfamily and a key transcription factor for adipogenesis. It is alsoinvolved in various physiological processes, such as cell proliferation,angiogenesis, inflammation and lipid partitioning (Tontonoz &Spiegelman, 2008). Combination therapies with TZDs have been shown todisplay beneficial effects on cancer cell death, while also leading to areduction of overall systemic toxicity to these chemotherapeuticregimens (Girnun et al, 2008; Girnun et al, 2007; Tikoo et al, 2009).However, the detailed molecular basis underlying the beneficial effectsof TZDs to platinum treatment has yet to be documented prior to thepresent invention.

In the tumor microenvironment, both stromal and cancer cells contributeto various types of extracellular matrix (ECM) proteins to activelyremodel the microenvironment favorably for tumor growth and metastasis.Such ECM proteins include fibronectin, laminin, collagen I (COL1),collagen IV (COL4) and collagen VI (COL6), and these ECM components aremarkedly modulated in response to chemotherapy (Dangi-Garimella et al,2011; Sherman-Baust et al, 2003; Su et al, 2007). They have beensuggested to cause drug resistance in solid tumors, including small-celllung cancer, ovarian cancer, pancreatic cancer and breast cancer(Heileman et al, 2008; Rintoul & Sethi, 2001; Sherman-Baust et al, 2003;Shields et al, 2012) through multiple pathways. These include aninduction of anti-apoptotic pathways (Sethi et al, 1999), decreased drugtransport (Netti et al, 2000) and increased survival signals, such asthose mediated through integrin-based pathways (Jean et al, 2011). COL6is composed of three alpha chains; α1, α2 and α3. Particularly, the α3chain of COL6 (COL6A3) has been highlighted as a promising candidatetriggering drug resistance against platinum-based therapeutics since itslevels are vastly increased in the cisplatin-resistant cancer cells invitro (Sherman-Baust et al, 2003; Varma et al, 2005). Nevertheless, themore detailed mechanism underlying how COL6A3 regulates drug-resistancehas remained elusive. Furthermore, compositions useful for inhibitingCOL6A3 have yet to be characterized in connection with chemotherapy.

SUMMARY OF THE INVENTION

Aspects of the present invention relate to methods and reagents forincreasing chemosensitivity to platinum-based chemotherapy. In oneaspect, a method of increasing chemosensitivity to platinum-basedchemotherapy is provided, comprising administering to a patient in needthereof an effective amount of an endotrophin-neutralizing agent. Theagent can be a monoclonal antibody, or fragment thereof, capable ofbinding to the C5 domain of the alpha3 chain of human collagen VI (e.g.,SEQ ID NO: 5). In some embodiments, the method can further includeadministering an effective amount of thiazolidinedione to said patient.

In a further embodiment there is provided a method of treating a cancerpatient comprising administering an effective amount of anendotrophin-neutralizing agent (e.g., an antibody that binds that bindsto the C5 domain of the alpha3 chain of human collagen VI) or TGFβantagonist (e.g., an antibody that binds to TGFβ). In some aspects, sucha method is further defined as a method for increasing chemosensitivityto platinum-based chemotherapy or for inhibiting angiogenesis in thepatient. In certain aspects, the patient is a cancer patient, such as apatient having a breast or colon cancer.

In further aspects, a method of the embodiments further comprisesadministering at least a second anti-cancer therapy to a patient. Forexample, the second anti-cancer therapy can be administered before,after or essentially simultaneously with an endotrophin-neutralizingagent or a TGFβ antagonist. In some aspects, the second anti-cancertherapy is a chemotherapy, radiotherapy, gene therapy, surgery, hormonaltherapy, anti-angiogenic therapy or cytokine therapy. In preferredaspects, the chemotherapy comprises a platinum-based chemotherapy, suchas cisplatin, oxaliplatin or carboplatin. In still further aspects, amethod of the embodiments further comprises administeringthiazolidinedione to the patient (e.g., in conjunction with aplatinum-based chemotherapy).

In further aspects, a patient for treatment according to the embodimentsis a patient who has been determined to express an elevated level ofendotrophin relative to control patient. For example, in some cases, apatient can be determined to have an elevated level of endotrophin bymeasuring the level an endotrophin polypeptide in a sample from thepatient such as a serum, stool or biopsy sample. In still furtheraspects, an elevated level of endotrophin can be determined by measuringan elevated level of COL6A3 RNA in a sample (e.g., a cancer cell sample)of the patient.

A further embodiment of the present invention includes a monoclonalantibody, or fragment thereof, capable of binding to the C5 domain ofthe alpha3 chain of human collagen VI (SEQ ID NO: 5) or the C5 domain ofthe alpha3 chain of mouse collagen VI (SEQ ID NO: 6). In some aspects,the antibody competes for binding of the C5 domain of the alpha3 chainwith the 10B6 monoclonal antibody. In certain embodiments, themonoclonal antibody, or fragment thereof, includes: a) a light chaincomprising three light chain complementary regions (CDRs) having thefollowing amino acid sequences: i) the light chain CDR1: QNINKY (SEQ IDNO: 7); ii) the light chain CDR2: NTN; iii) the light chain CDR3:LQHSSLYT (SEQ ID NO: 8); and a light chain framework sequence from animmunoglobulin light chain; and b) a heavy chain comprising three heavychain complementary regions (CDRs) having the following amino acidsequences: i) the heavy chain CDR1: GYTFTSYE (SEQ ID NO: 9); ii) theheavy chain CDR2: IYPESGST (SEQ ID NO: 10); iii) the heavy chain CDR3:TRGLRVLGYVMDV (SEQ ID NO: 11); and a heavy chain framework sequence froman immunoglobulin heavy chain. In some embodiments, the monoclonalantibody, or fragment thereof may include: i) the light chain variableregion with the amino acid sequence of SEQ ID NO: 2; and ii) the heavychain variable region with the amino acid sequence of SEQ ID NO: 4. Insome aspects, an antibody of the embodiments is a recombinant and/orpurified antibody. For example, the recombinant antibody can be a human,humanized antibody or de-immunized antibody. In still further aspects,the antibody is an IgG, IgM, IgA or an antigen binding fragment thereof.In yet further aspects, the antibody is a Fab′, a F(ab′)2, a F(ab′)3, amonovalent scFv, a bivalent scFv, or a single domain antibody.

In still further aspects, an antibody (or fragment thereof) inaccordance with the embodiments is conjugated or fused to an imagingagent or a cytotoxic agent. For example, the imagining agent can be anMM contrast agent, a radionuclide or a fluorescence moiety. In certainaspects, the antibody is conjugated to a chemotherapeutic agent such asa platinum-base chemotherapeutic. In still further aspects, an antibodycan be fused to a toxin moiety such as gelonin, granzyme or a bacterialtoxin. Such antibody conjugates and fusions can likewise be employed inthe methods of the embodiments.

In yet a further embodiment there is provided a pharmaceuticalcomposition comprising an antibody of the embodiments.

Another aspect of the invention relates to a method of treating ametabolic disorders-related disease (e.g., diabetes), comprisingadministering to a patient in need thereof an effective amount of anendotrophin-neutralizing agent. The agent can be a monoclonal antibody,or fragment thereof, capable of binding to the C5 domain of the alpha3chain of human collagen VI (e.g., SEQ ID NO: 5).

In some embodiments, endotrophin, a cleavage product of COL6A3 isidentified as being actively involved in mammary tumor progressionthrough enhancing the epithelial-mesenchymal transition (EMT), fibrosisand chemokine activity, thereby recruiting stromal cells to the tumormicroenvironment. Notably, all of these activities are associated withacquired drug resistance. In this study, increased levels of endotrophinfollowing cisplatin exposure are reported. This causescisplatin-resistance through enhancing the EMT. Furthermore, endotrophinlevels were decreased by combination therapy with TZD, leading to adecrease of EMT, fibrosis and vasculature, thereby enhancing cisplatinsensitivity. Taken together, these results suggest that the beneficialeffects of TZDs on platinum-based chemotherapy are mediated through theinhibition of endotrophin in mammary tumors, and that the neutralizationof endotrophin activity is a key determinant to unleash the fullbeneficial effects of TZDs.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1A-1G: Expression profiles of ETP. (FIG. 1A) COL6 is composed ofCOL6α1, -α2, and -α3 chains. The C5 domain of the α3 chain, cleaved offthe COL6 microfilament, is highlighted in red. Shown are amino acidsequences compared between the human (SEQ ID NO: 5) and mouse (SEQ IDNO: 6) COL6α3-C5 domain. Conserved sequences are highlighted in yellow.(FIG. 1B) Western blots showing abundant secretion of ETP fromadipocytes. Conditioned media from 3T3-L1 preadipocytes and fullydifferentiated adipocytes were subjected to Western blotting usingα-mETP and α-COL6. Arrows indicate the secreted form of ETP. (FIG. 1C)ETP immunostaining of mammary gland tissues from obese animals, ob/oband db/db mice, compared with lean controls (n=5 per group). Arrowsindicate crown structure. Scale bars: 50 μm. (FIG. 1D and FIG. 1E)holo-COL6 and ETP immunostaining of tumor tissues from 9-week-old PyMTmice with α-COL6 (FIG. 1D) and α-mETP antibody (FIG. 1E), respectively.Lung tissues from 13-week-old PyMT mice were used for metastasized tumorlesions (FIG. 1F). Scale bars: 50 μm. (FIG. 1G) α-COL6- andα-ETP-positive staining area in tumor or stroma for the primary tumors.Data are mean±SEM of multiple fields in n=5 per group. ***P<0.001 vs.tumor, unpaired t test.

FIGS. 2A-2D: ETP levels in human cancer specimens and its targettissues. (FIG. 2A and FIG. 2B) Human cancer tissues compared with thoseof benign tissues were immunostained with human ETP-specific polyclonalantibody. Human samples for breast cancer (FIG. 2A) and colon cancer(FIG. 2B) were analyzed. Scale bars: 25 (FIG. 2C and FIG. 2D) Whole bodyin vivo imaging of injected ETP. IRD-800 fluorescence-labeled ETPprotein (10 μg) was intravenously injected into WT, 8-week-old PyMT, and10-week-old PyMT mice by tail vein. (FIG. 2C) ETP levels were visualizedby the Licor Infrared Scanner 10-90 minutes after injection. Arrowsindicate mammary tumors. L, liver; B, bladder; T, tumor. IgG was used asa negative control. (FIG. 2D) Tissues were excised 2 hours afterinjection, and ETP-positive fluorescence signals were determined with aLicor Infrared Scanner. Quantified values were normalized to total areaand represented as percentage of WT. ***P<0.001.

FIGS. 3A-3E: Transgenic mice expressing ETP under the MMTV promoter.(FIG. 3A) ETP immunostaining showing strong ETP positive signal inmammary ductal epithelium in the transgenic mice lines (ETP low andhigh). Scale bars: 50 (FIG. 3B and FIG. 3C) Antiapoptotic effects ofETP. (FIG. 3B) Apoptosis for mammary epithelial cells during involutionwas determined by TUNEL assay on mammary glands of WT, ETP, and mice 2days after forced weaning. Arrows indicate TUNEL-positive apoptoticcells. Scale bars: 50 (FIG. 3C) Quantification of TUNEL-positive cells,represented as mean±SEM (multiple images, n=3 per group). ***P<0.001,*P<0.05 vs. WT, unpaired t test. (FIG. 3D and FIG. 3E) Promitoticeffects of ETP. High ETP expressers (32 weeks old) spontaneouslydeveloped tumors. (FIG. 3D) Whole body image (left; arrows indicatetumors) and H&E staining of mammary gland (middle) and lung (right)tissue. Boxed regions are shown at higher magnification below. Scalebars: 200 μm. (FIG. 3E) Cell proliferation was determined by Ki67staining with mammary glands of 32-week-old ETP high-expressing and WTmice. Scale bars: 50 μm.

FIGS. 4A-4F: ETP augments primary tumor growth and pulmonary metastasisin the background of PyMT mice. (FIG. 4A) ETP immunostaining, with ahigh ETP-positive signal in tumor tissues from 12-week-old PyMT andPyMT/ETP mice. Scale bars: 50 Intensity of ETP staining was quantifiedand represented as mean±SEM (n=5 per group). *P=0.02 vs. PyMT, unpairedt test. (FIG. 4B) Whole-mount staining of mammary gland tissues from8-week-old PyMT and PyMT/ETP mice, with early neoplastic lesion areasincreased by ETP. (FIG. 4C) Tumor volume was determined by weeklycaliper measurements from PyMT (n=35) and PyMT/ETP (n=38) mice. Resultsare represented as mean±SEM. P=NS, 2-way ANOVA. (FIG. 4D and FIG. 4E)ETP augmented pulmonary metastasis. (FIG. 4D) Pulmonary metastaticgrowth was determined by measuring the tumor incidence in lung tissues(8- to 17-week-old, n=22-25 per group). H&E-stained preparations forlung tissues were used for analysis. Shown is percent metastasis-freemice over time. *P=0.025, log-rank test. (FIG. 4E) Representative H&Estain for lung tissues showing the degree of pulmonary metastasis in 15-and 17-week-old PyMT and PyMT/ETP mice. (FIG. 4F) Representativewhole-body images for tumor burden. Tumor volume for 13-week-oldFP635/PyMT and FP635/PyMT/ETP mice was monitored by IVIS fluorescencescanner. Metastatic burden was determined by fluorescence signals inlung tissues. Quantified results are represented as mean±SEM (n=8-9 pergroup). *P=0.0117, **P=0.0011 vs. PyMT, unpaired t test.

FIGS. 5A-51I: Histological analysis for tumor tissues of PyMT/ETP versusPyMT mice. (FIG. 5A) Proliferation indices were determined byimmunostaining with Ki67. Quantified results represent mean±SEM (n=5 pergroup). P=NS vs. PyMT, unpaired t test. (FIG. 5B) Fibrosis indices weredetermined by Masson's Trichrome C stain. Percent fibrotic area over thetumor lesions was quantified. Data represent mean±SEM (n=5 per group).**P=0.01 vs. PyMT, unpaired t test. Arrows indicate collagen fibrils.(FIG. 5C) Functional blood vessel areas were determined by lectinperfusion. Podoplanin (lymphangiogenesis marker) and DAPI (nucleus) werecostained. Quantified results represent mean±SEM (n=5 per group).**P=0.003 vs. PyMT, unpaired t test. (FIG. 5D) Hypoxia was determined bypimonidazole-HCl injection. Hypoxic lesions were stained in dark brown.Quantified results represent mean±SEM (n=5 per group). ***P=0.0007 vs.PyMT, unpaired t test. (FIGS. 5E-5H) Total RNA was prepared from thetumor tissues from PyMT/ETP and PyMT mice. mRNA levels for the genesresponsible for fibrosis and EMT (FIG. 5E and FIG. 5F), angiogenesis andlymphangiogenesis (FIG. 5G), and inflammation (FIG. 5H) were determinedby qRT-PCR. mRNA levels were normalized with β-actin and represented asmean±SEM (n=8 per group). Relative values of each gene are representedas fold change relative to PyMT. *P<0.05, ***P<0.001 vs. PyMT, 2-wayANOVA. Scale bars: 50 μm (FIGS. 5A-5C); 100 μm (FIG. 5D). Insets in FIG.5A are enlarged ×5.

FIGS. 6A-61I: ETP augments metastasis through enforcing TGF-β-dependentEMT. (FIG. 6A) E-cadherin immunostaining for tumor tissues from PyMT andPyMT/ETP. (FIG. 6B and FIG. 6C) SBE-luciferase reporter assay. SeeSupplemental Methods for details. Data represent fold increase (3independent experiments). **P<0.01, *P<0.05, 2-way ANOVA. pRA-ctrl,empty; pRA-sETP, secretion form; pRA-ETP, intracellular form. (FIGS.6D-6G) Allografts of Met-1 cells in the presence of either ETP (20ng/plug) or PBS mixed with 1D11 or IgG (10 μg/plug) within a Matrigelplug. 10 days after implantation, additional 1D11 or IgG (100 μg) wasi.p. injected once a week during tumor progression. (FIG. 6D) Tumorvolumes represent means±SEM (n=5 per group). *P<0.05, 2-way ANOVA. (FIG.6E) H&E staining. The ratio of stromal area in tumor tissues wasquantified. Data represent mean±SEM (n=5 per group). *P<0.05, unpaired ttest. T; tumor and S; stroma. (FIG. 6F) Fibrosis was determined byMasson's Trichrome C stain. Data represent mean±SEM (n=5 per group).**P<0.01, ***P<0.001, unpaired t test. (FIG. 6G) Western blotting forEMT markers E-cadherin, vimentin, and α-SMA. β-actin, loading control.Data represent fold increase (n=5 per group). *P<0.05, **P<0.01,***P<0.001, unpaired t test. (FIG. 6H) Control and ETP⁺-cancer cellswere isolated from FP635/PyMT and FP635/PyMT/ETP mice and conveyed intoWT mice by tail vein injection (0.5×10⁶ cells/mouse). Either IgG or 1D11(100 μg) was i.p. injected every 5 days. 20 days post injection,metastasized cancer cells in the lung tissues were determined byfluorescence intensity. Data represent fold increase (n=3-4 per group).**P<0.01, *P<0.05, unpaired t test. Scale bars: 20 μm (FIG. 6A); 50 μm(FIG. 6E); 100 μm (FIG. 6F).

FIGS. 7A-7I: ETP acts as a chemokine augmenting tumor growth. (FIG. 7A)Tumor growth significantly increased in ETP⁺-tumor tissue allograft intoWT mice. Tumor volume was determined 1 month after implantation(representative images). Data represent mean±SEM (n=5 per group).***P<0.001, unpaired t test. (FIG. 7B) In vivo Matrigel bioassay.Matrigel (50 μl) was mixed with ETP (100 ng/plug) or PBS and implantedinto WT mice in the presence of IgG, 1D11, or 10B6 (20 μg/plug). 2 daysafter implantation, plugs were excised and stained for FIG. 7H & FIG.7E. Scale bars: 100 μm. (FIGS. 7C-7H) Cancer cells were plated in thebottom chamber 1 day prior to seeding MS-1 cells (5×10⁵ cells/well) andmacrophages (1×10⁵ cells/well) atop the membrane chamber in Transwelland incubated for 18-24 hours (FIG. 7C). (FIGS. 7D, 7E, 7F, 7G)Representative images of multiple independent experiments. Scale bars:100 μm. (FIG. 7H) Quantitation (mean±SEM; n=3 per group). ***P<0.001,**P<0.05, *P<0.01, unpaired t test. (FIG. 7I) MS-1 migration assay. MS-1cells (5×10⁵ cells/well) were plated atop the chamber in Transwell andincubated for 24 hours. Chemotaxis was set up by following cellmigration from DMEM/serum-free to DMEM/2% FBS/PBS or DMEM/2% FBS/ETPprotein (1 μg/well). Images are representative of multiple independentexperiments. Data represent mean±SEM (n=3 per group). ***P=0.008,unpaired t test. Scale bars: 100 μm.

FIGS. 8A-8I: 10B6 blocks ETP-mediated stromal expansion in vivo. (FIG.8A) 10B6 (200 μg/mouse) was i.p. injected twice weekly into PyMT micefrom 9 to 13 weeks of age. Tumor growth (mean±SEM; n=4-6 per group) wasdetermined by weekly caliper measurements. *P<0.05, **P<0.01, ***P<0.001vs. IgG control, 2-way ANOVA. (FIG. 8B) Primary mammary epithelialcancer cells were isolated from 12-week-old FP635/PyMT andFP635/PyMT/ETP mice and implanted into WT recipients with the samevolume of Matrigel. For the 10B6 group, 10B6 was added in a Matrigelplug (10 μg/plug) admixed with ETP⁺-cancer cells (i.e., ETP⁺/10B6). Arepresentative whole-body image was acquired 25 days after implantationusing IVIS fluorescence scanner. Artificial color indicates fluorescencesignal intensity accounts for tumor volume (AU). Quantitative resultsare represented as mean±SEM (n=3 per group). *P<0.05, unpaired t test.(FIGS. 8C-8I) 6 weeks after implantation, tumor tissues were excisedfrom Ctrl-, ETP⁺-, and ETP⁺/10B6-cancer cells allografted mice andstained for H&E (FIG. 8C), Masson's Trichrome C (FIG. 8D), α-SMA (FIG.8E), FSP-1 (FIG. 8F), CD31 (FIG. 8G), F4/80 (FIG. 8H), and Ki67 (FIG.8I. Quantified results in FIGS. 8D-8I are mean±SEM (multiple images, n=3per group). *P<0.05, **P<0.01, unpaired t test. Scale bars: 50 μm. (FIG.8J) Working model for ETP in mammary tumor progression.

FIGS. 9A-9C. Generation of endotrophin-specific antibodies. FIG. 9A.HEK-293 cell produced mouse endotrophin was subjected to Westernblotting using rabbit anti-mouse endotrophin polyclonal antibody. FIG.9B. Bacterially produced GST-fused recombinant human endotrophin proteinwas subjected to western blotting using a rabbit anti-human endotrophinpolyclonal antibody. GST protein was used as a negative control. Thearrow points at endotrophin. FIG. 9C. Rat anti-mouse endotrophinmonoclonal antibodies efficiently capture the native form of endotrophinprotein. IRD (infrared dye)-800 labeled native form of endotrophinprotein was incubated with either endotrophin monoclonal antibodies,including 4D1, 4F8, 10B6-A11, 10B6-B5, and 10B6-C3 or a rat-IgG for 2hours at room temperature and subsequently incubated with Protein GSepharose for 1 hour. The protein-Sepharose complex was separated on a10-20% Tricine gel after 3 times washing with PBS. Captured endotrophinprotein was visualized on a Licor Odyssey Infrared Scanner (LicorBioscience). Green color represents the IRD800 channel.

FIGS. 10A-10F. Endotrophin expression levels in various tissues. FIGS.10A-10B. Endotrophin is highly expressed in human breast cancer (FIG.10A) and colon cancer (FIG. 10B) tissues compared to samples obtainedfrom benign lesions. Human breast and colon cancer samples (UTSW MedicalCancer Human Tissue Bank) were immunostained with polyclonal humanendotrophin antibodies (TX933). Human samples for breast- and coloncancer patients were analyzed. Scale bars: 100 μm (10×) and 25 μm (40×).FIG. 10C. COL6 immunostaining for adipose tissues of obese animals suchas ob/ob and db/db mice compared to lean control mice (pan-collagen 6antibody, Abcam, Ab6588). Scale bars: 50 μm. FIG. 10D. Endotrophin mRNAlevels in various cell lines: mRNA levels for endotrophin weredetermined by qRT-PCR with various cell lines such as MS1 (mouseendothelial cells), mouse primary macrophages, and Met-1 (mouse mammarycancer cells). qRT-PCR results were normalized with β-actin. FIGS.10E-10F. Tissue distribution of COL6. Various tissues were collectedfrom 10-week-old FVB WT mice or tumor tissues from PyMT mice andanalyzed for COL6A1, -A2, and -A3 mRNA levels. qRT-PCR results werenormalized with 18S RNA (FIG. 10E). **p<0.01, ***p<0.001 vs. mammarygland (M. gland) by 2-way ANOVA (n=4/group). Results were normalizedwith β-actin and represented as mean±SEM (FIG. 10F).

FIGS. 11A-11G. Generation of the MMTV-endotrophin transgenic mice. FIG.11A. Diagram for the MMTV-endotrophin transgenic mice. FIG. 11B. Tissuedistributions of endotrophin transgene. mRNA levels for the endotrophintransgene were determined by qRT-PCR with various tissues from lowexpressers (line 2145, left Y-axis) and high expressers (line 2246,right Y-axis). Results were normalized with β-actin. FIGS. 11C-11D.Mammary ductal epithelium growth develops normally in the endotrophintransgenic mice. Morphological analysis of ductal epithelial growth wasperformed with whole mount preparations (FIG. 11C) and H&E stain (FIG.11D) of inguinal mammary glands from 8-week-old WT, and endotrophintransgenic (ETP-Tg; low and high expresser) mice. Scale bars: 50 μm inH&E. FIG. 11E. Tissue fibrosis was determined by Masson's Trichrome Cstaining of 8-week-old mice. Collagen fibrils are stained with blue.Scale bars: 50 μm. Quantified results are represented as mean±SEM(n=5/group). p=n.s (no significance) vs. WT by unpaired t-test. FIG.11F. Reproduction was determined by measuring pregnancy incidence,litter size, and duration of pregnancy (from mating to delivery). n=5-9per group. FIG. 11G. The rate of involution was determined bymorphological analysis with H&E preparations of mammary gland atindicated days after forced weaning.

FIGS. 12A-12B. Quantification of tumor progression with theinfrared-fluorescence protein (FP635) transgenic mice driven by MMTVpromoter. FIG. 12A. Infrared fluorescence protein (FP635) is exclusivelyexpressed in the mammary ductal epithelium under the control of MMTVpromoter. A MMTV-FP635 transgenic mouse line was established startingwith 7 independent founders by screening for FP635 fluorescence proteinexpression in frozen sectioned mammary gland tissues. Images wereacquired using a Leica confocal microscope. The DAPI stain highlightsnuclei. FIG. 12B. Longitudinal whole body in vivo tumor imaging withMMTV-FP635 transgenic mice (FP635/PyMT). Female MMTV-FP635 mice werecrossed with male MMTV-PyMT mice to obtain female FP635/PyMT mice. Tumorvolume for the PyMT/FP635 mice at indicated time points was determinedby integration of infrared fluorescence signal expressed in the ductalepithelium during tumor progression. Images were acquired with a MaestroFluorescence Scanner.

FIGS. 13A-13B. Gene expression profiling for tumor tissues fromPyMT/endotrophin compared to PyMT. FIG. 13A. cDNA microarrays for tumortissues from PyMT vs. PyMT/ETP were analyzed. Diagram represents % ofmodulated genes by PyMT/ETP vs. PyMT. Functional annotation for genessignificantly changed by endotrophin is represented as a bar-graph.Analysis was performed with DAVID Bioinformatics Resources 6.7, NationalInstitute of Allergy and Infectious Diseases (NIAID), NIH(http://david.abcc.ncifcrf.gov/home.jsp). FIG. 13B. Canonical pathwayanalysis for the cDNA microarray data was performed with IngenuitySystem (http://www.ingenuity.com). Top 5 ranked canonical pathways arerepresented as a bar graph.

FIGS. 14A-14B. Generation of mammalian cell-produced endotrophin. Mouseendotrophin cDNA in the presence of either a 5′ in-frame prolactinsignal sequence (sETP) or an adiponectin signal sequence (ETP) wascloned into the mammalian expression vector pRA-GFP. Each construct wastransiently transfected into HEK293 cells and supernatants werecollected 2 days post transfection. FIG. 14A. Supernatants weresubjected to SDS-PAGE and Coomassie staining to check for endotrophinsecretion. FIG. 14B. Cell lysates and supernatants were subjected towestern blotting with anti-mouse endotrophin antibody. Intracellularendotrophin was presented in both ETP- and sETP-expressing cells.Whereas only sETP-overexpressing cells secrete endotrophin into themedia, since the adiponectin signal sequence is an inefficient signalfor secretion of a passenger protein. Arrow indicates secretedendotrophin.

FIGS. 15A-15D. Endotrophin has a limited effect on tumor cellproliferation. FIG. 15A. Endotrophin⁺-cancer cells grow at comparablerates as ctrl-cancer cells. An equal number of cancer cells were freshlyisolated from tumor tissues of PyMT (ctrl-cancer cells) andPyMT/endotrophin (ETP⁺-cancer cells) and implanted into WT mice (1×10⁶cells/mouse). Tumor volume was determined 1 month post-implantation.Quantitative results are represented as mean±SEM (n=7/group). p=n.s vs.Ctrl-cancer cells by unpaired t-test. FIG. 15B. Met-1 cells were seededinto the 96 well plates and incubated with DMEM/10% FBS/PBS, DMEM/10%FBS/endotrophin, or conditioned media acquired from HEK293 orHEK293/endotrophin-overexpressing cells for 24 hours. Mitotic activitywas measured with a Mitotic Index Assay Kit (Active Motif) following themanufacturer's protocol. Paclitaxel (1 μM) was used as a positivecontrol of mitosis. Quantified results are represented as mean±SEM(n=10/group) p=n.s vs. PBS or Ctrl-media by unpaired t-test. FIG. 15C.Human breast cancer cells, MCF7 (0.1×10⁵) were plated in the 24 wellplates with or without recombinant endotrophin protein and cell numberswere counted over time. The quantification of results is represented asmean±SEM (n=6/group, triplicate). p=n.s vs. PBS by unpaired t-test. FIG.15D. Tumor tissues taken from FIG. 15A were immunostained forendothelium with anti-endomucin antibody (Santa Cruz. Biothechnology,Inc., sc-65495), demonstrating that the vascularization is significantlyincreased in ETP⁺-cancer cells compared to Ctrl-cancer cells. Endomucinpositive area was quantified and represented as mean±SEM (multipleimages, n=4/group). ***p=0.0008 vs. ctrl by unpaired t-test. Scale bars:200 μm.

FIGS. 16A-16J. Endotrophin augments tumor angiogenesis, fibrosis andinflammation through mediating tumor-stromal interactions. Small piecesof tumor tissues derived from 12-week-old PyMT (Ctrl-tumor) andPyMT/endotrophin (ETP⁺-tumor) were implanted into the left or right sideof a fat pad of a WT recipient, respectively. FIGS. 16A-16C.Angiogenesis is significantly increased in ETP⁺-tumors. Blood vesselarea was determined by immunostaining with CD31; Functional vessel areawas determined by lectin perfusion for Ctrl-tumors, tumor size adjustedCtrl (SA)-tumors and ETP⁺-tumors. DAPI (nucleus) was co-stained. Scalebars: 100 μm. CD31⁺-area for blood vessel area (FIG. 16B) and functionalvessel area (FIG. 16C) were quantified and represented as mean±SEM (5independent images, n=5/group). *p<0.05 vs. Ctrl (SA)-tumor by unpairedt-test. FIGS. 16D-16E. Hypoxia regions were determined with a hypoxiaprobe (pimonidazole-HCL). Quantified results are represented asmean±SEM. *p<0.005 vs. Ctrl (SA)-tumor by unpaired t-test. FIGS.16F-16G. Fibrosis indices were determined by Masson's Trichrome C stain.Scale bars: 50 μm. Quantified results are represented as mean±SEM.**p<0.005 vs. Ctrl (SA)-tumor by unpaired t-test. FIG. 16H.Immunostaining for α-SMA⁺ myofibroblasts. FIGS. 16I-16J. Immunostainingfor F4/80⁺ macrophages. DAPI (nucleus) was co-stained. Scale bars: 100μm. Quantified results are represented as mean±SEM. *p=0.03 vs. Ctrl(SA)-tumor by unpaired t-test.

FIGS. 17A-17B. Endotrophin effects on endothelial cell migration. FIG.17A. in vivo Matrigel bioassays were performed as described in FIG. 6J.Matrigel plugs were analyzed by H&E and immunostaining for CD31(endothelial cell marker). Scale bars: 200 μm (H&E), and 50 μm (CD31).FIG. 17B. in vitro Angiogenesis assay. MS-1 cells (5×10⁴ cells/well)were plated in triplicate on the matrigel coated 12-well plates. Tubuleformation was assessed 3 hours after incubation with conditioned mediaderived from H293 or H293-endotrophin overexpressing cells in thepresence or absence of 10B6 (10 μg/well). Scale bars: 500 μm.

FIG. 18. Contributions of stromal adipocytes to COL6 mRNA expressions.COL6A1-, A2 and -A3 mRNA levels in adipocytes (Adi) and stromal-vascular(S.V.) fractions in mammary glands for WT and PyMT mice. mRNA levelswere determined by RT-qPCR. Results were normalized to 0-actin andrepresented as mean±SEM. Bar graph is represented as -fold increase overthe S.V fraction. **p<0.01, ^(###)p<0.001 vs. S.V by unpaired studentt-test. (n=4/group, tissues from 3 mice were pooled in one sample; i.e.,total 12 mice/group).

FIGS. 19A-19F. TZD augments cisplatin sensitivity and correlates withthe COL6A3 levels. FIG. 19A. FP635/PyMT mice were given TZD containingchow (supply approx. 20 mg/kg/day, rosiglitazone) or normal-diet (ND)starting at 8-weeks of age, and cisplatin (1 mg/kg) or PBS treatment wasinitiated at 10 weeks of age (ip., 3 times/week) over the course oftumor progression. Tumor burden was monitored with a fluorescencescanner (IVIS, Caliper life science). Quantified results are representedas mean±SD (n=8-9/group). *p=0.04, ND/CIS vs. TZD/CIS by unpairedstudent t-test. Metastatic burden was determined by fluorescence signalsin lung tissues. FIG. 19B. Primary cancer cells isolated from tumors inPyMT mice were implanted into WT mice. TZD were given 5 days prior tocisplatin treatment (1 mg/kg, every 5 days). Tumor volumes weredetermined by caliper measurement and represented as mean±SD(n=5-6/group). *p<0.05 and **p<0.001, ND/CIS vs. TZD/CIS by 2-way ANOVA.FIGS. 19C-19D. Total RNA was extracted from tumor tissues in each group.mRNA levels for collagens such as COL1A1, COL6A1, -A2, and -A3 (FIG.19C), and EMT genes such as E-cadherin, N-cadherin, Vimentin, Snail,Slug, Twist1 and Twist2 (FIG. 19D) were determined by qRT-PCR andnormalized with 36B4. Quantitative results represent mean±SD(n=7/group). *p<0.05, **p<0.01, ***p<0.001 ND/PBS vs. ND/CIS;^(###)p<0.001 ND/CIS vs. TZD/CIS by 2-way ANOVA. FIGS. 19E-19F. EMTindices were determined by immunostaining with E-Cadherin (FIG. 19E) andVimentin (FIG. 19F). Cytokeratin (epithelial cells) and DAPI (nucleus)were co-stained. Staining positive area was quantified (multiple images,n=5/group). **p=0.014 (E) and *p=0.015 (F), ND/CIS vs. TZD/CIS byunpaired student t-test. Scale bars: 100 μm.

FIGS. 20A-20C. Endotrophin overexpression confers cisplatin resistancein PyMT mice. FIG. 20A. 10-weeks-aged PyMT and PyMT/endotrophin(PyMT/ETP) mice were given high dosage of cisplatin (2.5 mg/kg, ip., 2times/week). Tumor growth was determined by caliper measurements. Datarepresent mean±SD (n=7-10/group). ***p<0.001, PyMT/CIS vs. PyMT/ETP/CISby 2-way ANOVA. FIGS. 20B-20C. A piece of tumors taken from PyMT(Ctrl-tumor) and PyMT/endotrophin (ETP⁺-tumor) mice were implanted intoisogenic wild-type hosts. Cisplatin (1 mg/kg, ip., 2 times/week) wereinjected at 3-weeks post implantation for tumor progression. Tumorvolume was determined by caliper measurement. Quantification (FIG. 20B)and representative images (FIG. 20C) showing increased cisplatinresistance in ETP⁺-tumors. Data represent mean±SD (n=7-8/group). *p<0.05vs. Ctrl-tumors by 2-way ANOVA. Representative images were taken at70-days post implantation. Scale: 10 mm.

FIGS. 21A-21F. TZD synergizes cisplatin sensitivity through suppressionof endotrophin-mediated EMT, fibrosis and vasculature. FIGS. 21A-21B.Schematic diagram for allografts (FIG. 21A), indicating cancer cells(0.5×10⁶ cells/mouse) were isolated from tumors in PyMT (Ctrl) andPyMT/endotrophin (ETP) mice and implanted into wild-type hosts. Hostmice were given TZD (20 mg/kg) or ND diet at 10 days before implantationfor tumor progression. Cisplatin (1 mg/kg, ip., every 5 days) wasadministered at 3-weeks post implantation. Quantification of tumorvolume (FIG. 21B), showing TZD suppressed tumor growth inendotrophin⁺-tumors. Data represent mean±SD (n=8-9/group). *p=0.05,**p=0.01 and ***p=0.001 Ctrl/ND vs. ETP/ND; ^(##)p=0.01 and^(###)p=0.001ETP/ND vs. ETP/TZD by unpaired student t-test. FIGS. 21C-21F.Histological analysis of tumors in allografts after cisplatin treatment.H&E staining and necrotic area quantification (FIG. 21C), showingsignificantly increased chemo-sensitivity in endotrophin⁺ tumors uponcombination of TZD with cisplatin. Necrotic area (*). ***p<0.0001,and^(##)p=0.0018. Vimentin staining quantification (FIG. 21D), showingdecreased EMT in both Ctrl- and endotrophin⁺-tumors by TZD. ***p<0.0001and^(###)p<0.0001. Quantification of perfused lectin staining (FIG.21E), showing the increased functional blood vessels inendotrophin⁺-tumors was decreased by TZD. *p=0.015, **p=0.001 and^(##)p=0.0088. Masson's Trichrome C staining quantification (FIG. 21F),showing increased fibrosis in endotrophin⁺-tumors was decreased by TZD.***p<0.0001 and ^(##)p=0.0013. Scales: 200 μm (FIG. 21A), 100 μm (FIGS.21D-21E) and 50 μm (FIG. 21F). Statistics (*Ctrl/ND/CIS vs. Ctrl/TZD/CISor ETP/ND/CIS; ^(#)ETP/ND/CIS vs. ETP/TZD/CIS) were analyzed by unpairedstudent t-test.

FIGS. 22A-22E. Neutralizing endotrophin activities with monoclonalantibodies sensitizes tumors to cisplatin treatment. FIG. 22A. Pieces oftumors from PyMT mice were implanted into wild-type hosts. Tumor-bearingmice were given cisplatin (1 mg/kg, ip., every 5 days) or PBS, combinedwith either TZD (20 mg/kg) or anti-endotrophin monoclonal antibodies(100 μg/mouse, once a week) for tumor progression. Tumor volumes weredetermined by caliper measurements. Data represent mean±SD (n=5/group).*p<0.05, **p<0.01, ***p<0.001 vs. Cisplatin by 2-way ANOVA. FIGS.22B-22D. 4T1 (0.5×10⁶/mouse) cells were xenografted in nude mice andmonitored tumor growth (FIG. 22B) and metastasis (FIG. 22C). Cisplatin(1 mg/kg, every 5 days, i.p) with either 10B6 or IgG control (100μg/mouse, once a week, i.p) was given to tumor-bearing mice from 12-daysafter implantation. Tumor volumes were determined by calipermeasurements. Data represent mean±SD (n=5/group). *p<0.05, ***p<0.001vs. Cisplatin+IgG by 2-way ANOVA. Metastatic burden was determined bymeasuring metastatic lesion area in lung tissues with H&E stains.Quantified data represent mean±SD (n=5/group). **p=0.0007 and *p=0.0209vs. CIS/10B6 by unpaired student t-test. mRNA levels for EMT markerssuch as vimentin, snail, slug, twist1, twist2 and S100S4 were determinedby RT-qPCR (FIG. 22D). Values are normalized with 36B4 and representedas mean±SD (n=5/group). **p=0.0070, **p=0.0045, **p=0.0022 for vimentin,twist1, and S100A4, respectively by unpaired student t-test. FIG. 22E.Summary of study. Increased endotrophin following cisplatin treatmentconfers cisplatin resistance, and beneficial effects of TZDs oncisplatin sensitivity are mediated through both a suppression ofendotrophin levels and its downstream pathways, including EMT, fibrosisand angiogenesis.

FIGS. 23A-23B: TZD confers beneficial effects on cisplatin treatment inmammary tumors. Met-1 cancer cells (0.5×10⁶ cells/mouse) were implantedinto wild-type mice and TZD (20 mg/kg) was given by diet and cisplatin(1 mg/kg) was intraperitoneally (ip.) injected every 5 days from 30 dayspost implantation (n=8/group). ***p<0.001 vs. ND by 2-way ANOVA. Tumortissues from PyMT mice given TZD or normal diet (ND) diet werehistologically assessed. Representative images and quantification forTUNEL staining (FIG. 23A), showing increased cancer cell apoptosis byTZD combination in cisplatin. Data represent mean±SD (n=5/group).**p=0.0042 ND vs. TZD by unpaired student t-test. Scales: 50 μm.Representative metallothioneins staining and quantification (FIG. 23B),showing a higher signal in tumor tissues following cisplatin, which isefficiently suppressed by TZD combination. Data represent mean±SD(n=5/group). *p=0.002 ND vs. TZD by unpaired student t-test. Arrowsindicate staining positive cells. Scales: 25 μm.

FIGS. 24A-24D: Endotrophin neutralization improves the metabolicphenotype. FIG. 24A. Two cohorts of mice (n=8) were exposed to high fatdiet for 45 days after weaning. At the indicated time points(arrowheads), mice were treated with anti-endotrophin monoclonalantibodies (100 μg/mouse) or equivalent amounts of non-immune antibody.Body weights were monitored. FIG. 24B. Glucose infusion rate and FIG.24C. Suppression of hepatic glucose efflux was measured in ahyperinsulinemic euglycemic clamp. FIG. 24D. Table of parametersmeasured prior and during the clamp. Significant differences asindicated by unpaired student t-test.

FIG. 25: Endotrophin is upregulated in obese, insulin resistant humanfat tissue, but not in obese insulin sensitive fat tissue. Humanmesenteric adipose tissue biopsies from healthy obese and insulinresistant obese patients were immune-decorated with an anti-humanendotrophin antibody.

FIGS. 26A-26G: Endotrophin neutralization improves the metabolicphenotype. FIG. 26A. Two cohorts of mice (n=8) were exposed to high fatdiet for 45 days after weaning. At the indicated time points(arrowheads), mice were treated with anti-endotrophin monoclonalantibodies (100 □g/mouse) or equivalent amounts of non-immune antibody.Body weights were monitored. FIG. 26B. Plasma Triglycerides and FIG.26C. plasma free fatty acids (NEFAs) and FIG. 26D. plasma cholesterolwere measured. FIG. 26E. Hepatic triglycerides and FIG. 26F. hepaticcholesterol were measured. Significant differences as indicated byunpaired student t-test. FIG. 26G. Hepatic histology (H&E stain) clearlyreveals differences in hepatic lipid accumulation. Scale bar=25 μm.

FIG. 27: Endotrophin-mediated changes in the tumor stroma.Adipocyte-derived COL6A3 levels are increased during obesity.Endotrophin is cleaved from the COL6A3 parent chain within the tumormicroenvironment. Endotrophin potentiates TGFβ-dependentepithelial-mesenchymal transition (EMT) and fibrosis and displayschemoattractive activity, recruiting endothelial cells and macrophages,leading to enhanced angiogenesis and chronic inflammation. All of theseactivities induced by endotrophin synergistically lead to enhanced tumorgrowth and metastasis. Either the endotrophin neutralizing monoclonalantibody (10B6) or the TGFβ antagonizing monoclonal antibody (1D11)differentially attenuate a subset of endotrophin effects. EndoRc:endotrophin receptor.

FIGS. 28A-28D: 10B6 mAb information. FIG. 28A: 10B6 rat hybridoma cellline expressing an anti-mouse endotrophin monoclonal antibody. FIG. 28B:transient expression of cloned 10B6 antibody as a rat/human chimera inHEK293 cells. FIG. 28C: 10B6 Kappa chain sequence (coding DNA (SEQ IDNO: 1) and amino acid (SEQ ID NO: 2) sequences) and alignment withIGKV22S7*01 (SEQ ID NO: 12). FIG. 28D: 10B6 heavy chain sequence (codingDNA (SEQ ID NO: 3) and amino acid (SEQ ID NO: 4) sequences) andalignment with IGHV1S12*01 (SEQ ID NO: 13).

DETAILED DESCRIPTION

Collagen VI (COL6, encoded by the COL6A1, COL6A2, and COL6A3 genes) isan extracellular matrix protein that forms a microfilamentous network invarious connective tissues, including skeletal muscle, cartilage, skinand adipose tissue. Among the various tissues, adipose tissue is by farthe most abundant source of COL6 microfilaments. Clinically, mutationsin COL6 develop mild muscle myopathies (such as Bethlem myopathy andUllrich congenital muscular dystrophy), with symptoms of muscle weaknessand apoptosis combined with joint hyperlaxity and contracture. Agenetically engineered mouse model, deficient in COL6 microfilamentformation and secretion, has been widely used to investigate the rolesof COL6 under physiological and pathological conditions. COL6 deficiencyin mice leads to the development of muscle dystrophies resemblingBethlem myopathy in man. In the area of tumor biology, COL6 has beenidentified as a tumor-promoting factor abundantly produced and releasedfrom adipocytes. Subsequent analysis of the COL6 functional null micebred into the murine MMTV-PyMT mammary tumor model (mouse mammary tumorvirus-polyoma middle T antigen) showed a significant attenuation ofearly onset mammary tumor progression. Specifically, thecarboxyl-terminal domain of the COL6A3 chain is massively upregulated inthe malignant tumors of human patients compared to the remaining part ofCOL6A3 chain. As follow-up analysis demonstrated, the cleavage productfrom the carboxyl-terminus of the COL6A3 chain (referred to asendotrophin) accounts for the tumor-promoting effects associated withCOL6. Ectopic expression of the isolated endotrophin fragment within thetumor microenvironment of MMTV-PyMT mice drives an increase of bothprimary tumor growth and pulmonary metastasis through an enhancement ofthe expansion of the tumor stroma. Additional prominent effectsassociated with endotrophin overexpression in the tumor stroma includean increase in fibrosis, angiogenesis and inflammation through increasedfibrogenesis, a stimulation of epithelial-mesenchymal transition (EMT)and chemokine activities; these are well-established stromal phenomenathat support aggressive traits of tumors (FIG. 27). Indeed, neutralizingmonoclonal antibodies against endotrophin suppress tumor growth andreduce metastatic growth in MMTV-PyMT mice. EMT of tumors conveysmetastatic traits and multiple drug resistances to cancer cells. Sinceendotrophin is a potent stimulator of EMT, it suggests that theneutralization of endotrophin may lend itself to enhancechemo-sensitivity in combination with conventional therapeutic regimens,though this remains to be directly shown.

Adipose tissue is a crucial organ for the maintenance of whole bodyenergy homeostasis, and also a major source of COL6. The roles of COL6in metabolic homeostasis were examined even without a tumor burden.Metabolic characterization of the COL6A1 functional null mice bred witha genetically obese animal model, the ob/ob mouse, reveals that COL6deficiency improves systemic metabolic profiles, including enhancedinsulin sensitivity and glucose metabolism. This is likely due to anumber of changes, but the reduced fibrotic stress commonly seen inhypertrophic adipose tissues in obese status is likely to be acontributing factor. COL6 is upregulated in obese and dysfunctionaladipose tissue, and anti-diabetic treatment regimens lead to asuppression of COL6 expression. Tumor lesions in the microenvironmentlead to a further local enrichment of endotrophin, either throughstimulation of syntheses and/or cleavage of endotrophin from the matureprotein, or through an induction of production within the tumor lesionsthemselves. As such, endotrophin is likely to constitute one of the riskfactors that mediate the more aggressive lesion growth and worseprognosis seen in patients with higher body mass indices (BMIs). Moreimportantly, it is likely that endotrophin plays a pro-fibrotic andpro-inflammatory role in a number of additional tissues, even in theabsence of a tumor challenge. This may be relevant for adipose tissues,liver and kidney, all tissues that are prone to fibrosis and chronicinflammation under pathological conditions. Therefore, inhibition ofendotrophin activity under such pathological conditions is likely to beassociated with clinical improvements.

Endotrophin (ETP) is the major mediator of the COL6-mediated tumoreffects. ETP augmented fibrosis, angiogenesis, and inflammation throughrecruitment of macrophages and endothelial cells. Moreover, ETPexpression was associated with aggressive mammary tumor growth and highmetastatic growth. These effects were partially mediated throughenhanced TGF signaling, which contributes to tissue fibrosis andepithelial-mesenchymal transition (EMT) of tumor cells. The resultshighlight the crucial role of ETP as an obesity-associated factor thatpromotes tumor growth in the context of adipocyte interactions withtumor and stromal cells.

In addition, relationship between thiazolidinediones (TZDs), endotrophinand cisplatin resistance was examined in the context of a mammary tumormodel. COL6A3 levels are significantly increased in response tocisplatin exposure in tumors. Endotrophin, in turn, causes cisplatinresistance. The effects of endotrophin can be bypassed by administeringTZDs in wild-type mice (leading to a downregulation of endotrophin).This sensitizes tumors to cisplatin partly through the suppression ofendotrophin-induced epithelial-mesenchymal transition (EMT). Therefore,the chemosensitization obtained with TZDs is achieved through adownregulation of endotrophin. Treatment with an endotrophinneutralizing monoclonal antibody in combination with cisplatincompletely inhibits tumor growth of allografts of MMTV-PyMT tumors.Combined, the data suggest that endotrophin levels are a strongprognostic marker for the effectiveness of the combination therapy ofTZDs with cisplatin. Furthermore, neutralization of endotrophin activitydramatically improves the therapeutic response to combination therapy.

I. DEFINITIONS

For convenience, certain terms employed in the specification, examples,and appended claims are collected here. Unless defined otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs.

As used herein, the following terms and phrases are intended to have thefollowing meanings:

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

As used herein, the term “about” means within 20%, more preferablywithin 10% and most preferably within 5%.

An “antibody” is an immunoglobulin molecule capable of specific bindingto a target, such as a carbohydrate, polynucleotide, lipid, polypeptide,etc., through at least one antigen recognition site, located in thevariable region of the immunoglobulin molecule. As used herein, the termencompasses not only intact polyclonal or monoclonal antibodies, butalso fragments thereof (such as Fab, Fab′, F(ab′)₂, Fv), single chain(ScFv), mutants thereof, naturally occurring variants, fusion proteinscomprising an antibody portion with an antigen recognition site of therequired specificity, humanized antibodies, chimeric antibodies, and anyother modified configuration of the immunoglobulin molecule thatcomprises an antigen recognition site of the required specificity.

“Antibody fragments” comprise only a portion of an intact antibody,generally including an antigen binding site of the intact antibody andthus retaining the ability to bind antigen. Examples of antibodyfragments encompassed by the present definition include: (i) the Fabfragment, having VL, CL, VH and CH1 domains; (ii) the Fab′ fragment,which is a Fab fragment having one or more cysteine residues at theC-terminus of the CH1 domain; (iii) the Fd fragment having VH and CH1domains; (iv) the Fd′ fragment having VH and CH1 domains and one or morecysteine residues at the C-terminus of the CH1 domain; (v) the Fvfragment having the VL and VH domains of a single antibody; (vi) the dAbfragment which consists of a VH domain; (vii) isolated CDR regions;(viii) F(ab′)₂ fragments, a bivalent fragment including two Fab′fragments linked by a disulfide bridge at the hinge region; (ix) singlechain antibody molecules (e.g. single chain Fv; scFv); (x) “diabodies”with two antigen binding sites, comprising a heavy chain variable domain(VH) connected to a light chain variable domain (VL) in the samepolypeptide chain; (xi) “linear antibodies” comprising a pair of tandemFd segments (VH-CH1-VH-CH1) which, together with complementary lightchain polypeptides, form a pair of antigen binding regions.

“Chimeric antibodies” refers to those antibodies wherein one portion ofeach of the amino acid sequences of heavy and light chains is homologousto corresponding sequences in antibodies derived from a particularspecies or belonging to a particular class, while the remaining segmentof the chains is homologous to corresponding sequences in another.Typically, in these chimeric antibodies, the variable region of bothlight and heavy chains mimics the variable regions of antibodies derivedfrom one species of mammals, while the constant portions are homologousto the sequences in antibodies derived from another. One clear advantageto such chimeric forms is that, for example, the variable regions canconveniently be derived from presently known sources using readilyavailable hybridomas or B cells from non human host organisms incombination with constant regions derived from, for example, human cellpreparations. While the variable region has the advantage of ease ofpreparation, and the specificity is not affected by its source, theconstant region being human, is less likely to elicit an immune responsefrom a human subject when the antibodies are injected than would theconstant region from a non-human source. However, the definition is notlimited to this particular example.

A “constant region” of an antibody refers to the constant region of theantibody light chain or the constant region of the antibody heavy chain,either alone or in combination. The constant regions of the light chain(CL) and the heavy chain (CH1, CH2 or CH3, or CH4 in the case of IgM andIgE) confer important biological properties such as secretion,transplacental mobility, Fc receptor binding, complement binding, andthe like. By convention the numbering of the constant region domainsincreases as they become more distal from the antigen binding site oramino-terminus of the antibody.

The term “heavy chain” as used herein refers to the largerimmunoglobulin subunit which associates, through its amino terminalregion, with the immunoglobulin light chain. The heavy chain comprises avariable region (VH) and a constant region (CH). The constant regionfurther comprises the CH1, hinge, CH2, and CH3 domains. In the case ofIgE, IgM, and IgY, the heavy chain comprises a CH4 domain but does nothave a hinge domain. Those skilled in the art will appreciate that heavychains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α,δ, ε), with some subclasses among them (e.g., γ1-γ4). It is the natureof this chain that determines the “class” of the antibody as IgG, IgM,IgA IgG, or IgE, respectively. The immunoglobulin subclasses (isotypes),e.g., IgG1, IgG2, IgG3, IgG4, IgA1, etc. are well characterized and areknown to confer functional specialization.

The term “light chain” as used herein refers to the smallerimmunoglobulin subunit which associates with the amino terminal regionof a heavy chain. As with a heavy chain, a light chain comprises avariable region (VL) and a constant region (CL). Light chains areclassified as either kappa or lambda (κ, λ). A pair of these canassociate with a pair of any of the various heavy chains to form animmunoglobulin molecule. Also encompassed in the meaning of light chainare light chains with a lambda variable region (V-lambda) linked to akappa constant region (C-kappa) or a kappa variable region (V-kappa)linked to a lambda constant region (C-lambda).

As used herein, “neutralize” and permutations thereof refer to an agentthat is capable of inhibiting (partially or completely), reducing orabolishing an activity of a target (e.g., endotrophin).

“Nucleic acid,” “nucleic acid sequence,” “oligonucleotide,”“polynucleotide” or other grammatical equivalents as used herein meansat least two nucleotides, either deoxyribonucleotides orribonucleotides, or analogs thereof, covalently linked together.Polynucleotides are polymers of any length, including, e.g., 20, 50,100, 200, 300, 500, 1000, 2000, 3000, 5000, 7000, 10,000, etc. Apolynucleotide described herein generally contains phosphodiester bonds,although in some cases, nucleic acid analogs are included that may haveat least one different linkage, e.g., phosphoramidate, phosphorothioate,phosphorodithioate, or O-methylphophoroamidite linkages, and peptidenucleic acid backbones and linkages. Mixtures of naturally occurringpolynucleotides and analogs can be made; alternatively, mixtures ofdifferent polynucleotide analogs, and mixtures of naturally occurringpolynucleotides and analogs may be made. The following are non-limitingexamples of polynucleotides: a gene or gene fragment, exons, introns,messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA,cRNA, recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, isolated RNA of any sequence,nucleic acid probes, and primers. A polynucleotide may comprise modifiednucleotides, such as methylated nucleotides and nucleotide analogs. Ifpresent, modifications to the nucleotide structure may be impartedbefore or after assembly of the polymer. The sequence of nucleotides maybe interrupted by non-nucleotide components. A polynucleotide may befurther modified after polymerization, such as by conjugation with alabeling component. The term also includes both double- andsingle-stranded molecules. Unless otherwise specified or required, anyembodiment of this invention that is a polynucleotide encompasses boththe double-stranded form and each of two complementary single-strandedforms known or predicted to make up the double-stranded form. Apolynucleotide is composed of a specific sequence of four nucleotidebases: adenine (A), cytosine (C), guanine (G), thymine (T), and uracil(U) for thymine when the polynucleotide is RNA. Thus, the term“polynucleotide sequence” is the alphabetical representation of apolynucleotide molecule. Unless otherwise indicated, a particularpolynucleotide sequence also implicitly encompasses conservativelymodified variants thereof (e.g., degenerate codon substitutions) andcomplementary sequences as well as the sequence explicitly indicated.Specifically, degenerate codon substitutions may be achieved bygenerating sequences in which the third position of one or more selected(or all) codons is substituted with mixed-base and/or deoxyinosineresidues.

The terms “peptide,” “polypeptide” and “protein” used herein refer topolymers of amino acid residues. These terms also apply to amino acidpolymers in which one or more amino acid residues is an artificialchemical mimetic of a corresponding naturally occurring amino acid, aswell as to naturally occurring amino acid polymers, those containingmodified residues, and non-naturally occurring amino acid polymers. Inthe present case, the term “polypeptide” encompasses an antibody or afragment thereof.

A “variable region” of an antibody refers to the variable region of theantibody light chain or the variable region of the antibody heavy chain,either alone or in combination. The variable regions of both the light(VL) and heavy (VH) chain portions determine antigen recognition andspecificity. VL and VH each consist of four framework regions (FR)connected by three complementarity determining regions (CDRs) also knownas hypervariable regions. The CDRs complement an antigen's shape anddetermine the antibody's affinity and specificity for the antigen. Thereare six CDRs in both VL and VH. The CDRs in each chain are held togetherin close proximity by the FRs and, with the CDRs from the other chain,contribute to the formation of the antigen-binding site of antibodies.There are at least two techniques for determining CDRs: (1) an approachbased on cross-species sequence variability (the Kabat numbering scheme;see Kabat et al., Sequences of Proteins of Immunological Interest (5thed., 1991, National Institutes of Health, Bethesda Md.)); and (2) anapproach based on crystallographic studies of antigen-antibody complexes(the Chothia numbering scheme which corrects the sites of insertions anddeletions (indels) in CDR-L1 and CDR-H1 suggested by Kabat; seeAl-lazikani et al. (1997) J. Molec. Biol. 273:927-948)). Other numberingapproach or scheme can also be used. As used herein, a CDR may refer toCDRs defined by either approach or by a combination of both approachesor by other desirable approaches. In addition, a new definition ofhighly conserved core, boundary and hyper-variable regions can be used.

Other terms used in the fields of recombinant nucleic acid technology,microbiology, immunology, antibody engineering, and molecular and cellbiology as used herein will be generally understood by one of ordinaryskill in the applicable arts.

II. ANTIBODIES AND MODIFICATIONS OF ANTIBODIES

In one embodiment, the antibody is a chimeric antibody, for example, anantibody comprising antigen binding sequences from a non-human donorgrafted to a heterologous non-human, human, or humanized sequence (e.g.,framework and/or constant domain sequences). Methods have been developedto replace light and heavy chain constant domains of the monoclonalantibody with analogous domains of human origin, leaving the variableregions of the foreign antibody intact. Alternatively, “fully human”monoclonal antibodies are produced in mice transgenic for humanimmunoglobulin genes. Methods have also been developed to convertvariable domains of monoclonal antibodies to more human form byrecombinantly constructing antibody variable domains having both rodent,for example, mouse, and human amino acid sequences. In “humanized”monoclonal antibodies, only the hypervariable CDR is derived from mousemonoclonal antibodies, and the framework and constant regions arederived from human amino acid sequences (see U.S. Pat. Nos. 5,091,513and 6,881,557, incorporated herein by reference). It is thought thatreplacing amino acid sequences in the antibody that are characteristicof rodents with amino acid sequences found in the corresponding positionof human antibodies will reduce the likelihood of adverse immunereaction during therapeutic use. A hybridoma or other cell producing anantibody may also be subject to genetic mutation or other changes, whichmay or may not alter the binding specificity of antibodies produced bythe hybridoma.

Methods for producing polyclonal antibodies in various animal species,as well as for producing monoclonal antibodies of various types,including humanized, chimeric, and fully human, are well known in theart and highly predictable. For example, the following U.S. patents andpatent applications provide enabling descriptions of such methods: U.S.Patent Application Nos. 2004/0126828 and 2002/0172677; and U.S. Pat.Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,196,265; 4,275,149;4,277,437; 4,366,241; 4,469,797; 4,472,509; 4,606,855; 4,703,003;4,742,159; 4,767,720; 4,816,567; 4,867,973; 4,938,948; 4,946,778;5,021,236; 5,164,296; 5,196,066; 5,223,409; 5,403,484; 5,420,253;5,565,332; 5,571,698; 5,627,052; 5,656,434; 5,770,376; 5,789,208;5,821,337; 5,844,091; 5,858,657; 5,861,155; 5,871,907; 5,969,108;6,054,297; 6,165,464; 6,365,157; 6,406,867; 6,709,659; 6,709,873;6,753,407; 6,814,965; 6,849,259; 6,861,572; 6,875,434; and 6,891,024,each incorporated herein by reference.

In further embodiments, antibody molecules, or fragments thereof may beused to target some marker on the surface of a target cell. The antibodyalone may serve as an effector of therapy or it may recruit other cellsto actually affect cell killing. The antibody may also be conjugated toa drug or toxin (e.g., chemotherapeutic, radionuclide, ricin A chain,cholera toxin, pertussis toxin, etc.) and thus may merely serve as atargeting agent.

In certain embodiments, are antibody conjugates. The conjugate can be,for example, a specific binding agent (such as an antibody) of theinvention conjugated to other proteinatious, carbohydrate, lipid, ormixed moiety molecule(s). Such antibody conjugates include, but are notlimited to, modifications that include linking it to one or morepolymers. In certain embodiments, an antibody is linked to one or morewater-soluble polymers. In certain such embodiments, linkage to awater-soluble polymer reduces the likelihood that the antibody willprecipitate in an aqueous environment, such as a physiologicalenvironment. In certain embodiments, a therapeutic antibody is linked toa water-soluble polymer. In certain embodiments, one skilled in the artcan select a suitable water-soluble polymer based on considerationsincluding, but not limited to, whether the polymer/antibody conjugatewill be used in the treatment of a patient and, if so, thepharmacological profile of the antibody (e.g., half-life, dosage,activity, antigenicity, and/or other factors).

In further embodiments, the conjugate can be, for example, a cytotoxicagent. Cytotoxic agents of this type may improve antibody-mediatedcytotoxicity, and include such moieties as cytokines that directly orindirectly stimulate cell death, radioisotopes, chemotherapeutic drugs(including prodrugs), bacterial toxins (e.g., pseudomonas exotoxin,diphtheria toxin, etc.), plant toxins (e.g., ricin, gelonin, etc.),chemical conjugates (e.g., maytansinoid toxins, calechaemicin, etc.),radioconjugates, enzyme conjugates (e.g., RNase conjugates, granzymeantibody-directed enzyme/prodrug therapy), and the like. In one aspect,the cytotoxic agent can be “attached” to one component of a bi-specificor multi-specific antibody by binding of this agent to one of thealternative antigen recognition sites on the antibody. As analternative, protein cytotoxins can be expressed as fusion proteins withthe specific binding agent following ligation of a polynucleotideencoding the toxin to a polynucleotide encoding the binding agent. Instill another alternative, the specific binding agent can be covalentlymodified to include the desired cytotoxin.

In additional embodiments antibodies, or fragments thereof, can beconjugated to a reporter group, including, but not limited to aradiolabel, a fluorescent label, an enzyme (e.g., that catalyzes acolorimetric or fluorometric reaction), a substrate, a solid matrix, ora carrier (e.g., biotin or avidin). The invention accordingly provides amolecule comprising an antibody molecule, wherein the moleculepreferably further comprises a reporter group selected from the groupconsisting of a radiolabel, a fluorescent label, an enzyme, a substrate,a solid matrix, and a carrier. Such labels are well known to those ofskill in the art, e.g., biotin labels are particularly contemplated. Theuse of such labels is well known to those of skill in the art and isdescribed in, e.g., U.S. Pat. No. 3,817,837; U.S. Pat. No. 3,850,752;U.S. Pat. No. 3,996,345 and U.S. Pat. No. 4,277,437, each incorporatedherein by reference. Other labels that will be useful include but arenot limited to radioactive labels, fluorescent labels andchemiluminescent labels. U.S. patents concerning use of such labelsinclude for example U.S. Pat. No. 3,817,837; U.S. Pat. No. 3,850,752;U.S. Pat. No. 3,939,350 and U.S. Pat. No. 3,996,345. Any of the peptidesof the present invention may comprise one, two, or more of any of theselabels.

III. EXAMPLES Example 1: Adipocyte-Derived Endotrophin PromotesMalignant Tumor Progression

Breast cancer is the most common malignancy found in women. Among anumber of risk factors, obesity ranks high and contributes significantlyto postmenopausal breast cancer risk. Epidemiological evidence supportsa tight association among obesity, cancer incidence, and mortality.Hence, the adipocyte, as a major constituent of the mammary tumorstroma, is a likely contributor to tumor growth. The interactionsbetween malignant epithelial cancer cells and the surrounding stromalcells have a profound impact on tumor physiology, including cell growth,survival, metastasis, and recurrence. Numerous studies have documentedcontributions of stromal cells to tumor growth, through factors releasedfrom tumor-associated macrophages, fibroblasts, and endothelial cells.However, less is known about adipocyte factors that dominate the tumormicroenvironment; such factors are either permissive or, in some cases,actively contributing to tumor cell growth.

The adipocyte is an established endocrine organ, secreting varioussignaling molecules—such as adipokines, chemokines, and extracellularmatrix (ECM) constituents—in response to nutritional or hormonalstimuli. Adipocyte-derived factors involved in tumor progression includeproteins such as adiponectin, leptin, TNF-α, monocyte chemotacticprotein-1 (MCP-1), IL-6, and ECM components that control tumor cellbehavior within the tumor microenvironment. Key signaling networksassociated with cell proliferation, angiogenesis, inflammation, andapoptosis are activated by adipokines; these include PI3K, ERK1/2,STAT3, and NF-κB. Such pathways are frequently activated in tumortissues.

Collagen type VI (COL6; encoded by Col6a1, Col6α2, and Col6α3) isubiquitously expressed throughout connective tissues, such as bloodvessels, muscle, lung, and skin. However, adipose tissue (AT) is themost abundant source of COL6. COL6 is a large collagenous glycoproteincomposed of 3 chains, α1, α2, and α3, that are intracellularly assembledfrom heterotrimeric monomers to tetramers. Once secreted into theextracellular space, COL6 tetramers associate into microfibrils.Subsequently, the carboxyterminal C5 domain of the α3 chain isproteolytically cleaved off from the COL6 microfibrils. However, thedetails of this cleavage event and the functional role of the cleavageproduct, the C5 domain, remain unknown, with the exception that the C5domain plays an important structural role for COL6 microfibrilformation. Adipocyte-derived COL6 is a tumor-promoting factor in thebackground of the mammary tumor virus—polyoma middle T antigen(MMTV-PyMT) mammary tumor mouse model (referred to herein as PyMT mice).Notably, the carboxyterminal domain of the COL6α3 chain is stable andhighly enriched in human breast cancer specimens compared withfull-length COL6α3. However, prior to this study, it remains unknownwhether the cleaved C5 fragment of the COL6α3 chain, referred to hereinas endotrophin (ETP), participates in mammary tumor progression.

The studies herein examined whether ETP regulates tumor cell growth andmetastasis on its own, independent of other COL6 subunits, or theremainder of the COL6α3 chain. It is widely appreciated that the ECMprovides mechanical and structural support within the microenvironment.In addition, ECM-derived proteolytic fragments can directly activatevarious signaling pathways, influencing events in neighboring cells thatexpress ECM receptors, such as integrins. To better define the role ofETP in tumor progression within the local tumor microenvironment,independent of the rest of the COL6 complex, transgenic mice that harborETP with a signal sequence was generated under the control of themammary epithelial specific MMTV promoter. MMTV-ETP transgenic mice werecharacterized either independently (referred to herein as ETP mice), inthe background of PyMT mice (PyMT/ETP mice), or with tumor implantationsinto isogenic mice. These mouse models were used in combination withspecific ETP neutralizing antibodies to evaluate their therapeuticpotential. The aim of the studies was to identify and define mechanismsresponsible for the effects of COL6 on tumor growth and metastasis andfurther establish which signaling pathways play critical roles mediatingthe potent ETP effects.

Results

ETP is Abundant in Tumor Tissues.

To further investigate a role of COL6 in tumor progression, particularlyin the context of ETP, polyclonal antibodies specific for either mouseor human ETP domains were generated (FIGS. 9A and 9B); a substantialdegree of conservation was preserved between the species (FIG. 1A).Similar to holo-COL6 levels, secreted ETP was readily identified inconditioned media of 3T3-L1 adipocytes, but not 3T3-L1 fibroblasts (FIG.1B). Consistent with this observation, high ETP levels in the AT ofobese animals were observed, such as ob/ob and db/db mice, compared withlean controls (FIG. 1C). Interestingly, ETP prominently accumulated inobesity-associated crown-like structures of AT (FIG. 1C, arrows),prominent structures in dysfunctional adipocytes in which infiltratingmacrophages mediate chronic inflammatory responses. In contrast, aholo-COL6-specific antibody primarily highlighted a signal at theperiphery of adipocytes (FIG. 10C). Immunostaining of tumor tissues fromPyMT mice with anti-holo-COL6 showed that entire tumor lesions weresurrounded by COL6 fibrils, with weaker staining observed in AT (FIGS.1D and 1G). Interestingly, cleaved soluble ETP freely diffused in themicroenvironment and accumulated on primary tumor lesions of PyMT micein a paracrine manner (FIGS. 1E and 1G). Of note, ETP was less prominenton metastasized tumors in the lung (FIG. 1F), which suggests that ETPlevels on tumor cells may critically depend on the presence of localadipocytes to supply ETP. Histological analysis of human breast tumortissues indicated that ETP was highly abundant on both epithelial cancercells and various stromal cells within the tumor microenvironment, witha much lower signal seen in benign tissues (FIG. 2A and FIG. 10A). Inthe mouse, ETP was highly expressed in the mammary epithelial cancercell Met-1, relative to other cell types, such as the endothelial cellline MS-1 or primary macrophages (FIG. 10D). This suggests that cancercells can express ETP, even though AT was the major source for COL6among various WT tissues and PyMT tumor tissue (FIGS. 10E and 10F). ETPoverexpression was not restricted to mammary cancer cells. Similarincreases in ETP in other tumor sections was observed, such as in humancolon cancers, which showed significantly higher ETP levels than thosein benign tissues (FIG. 2B and FIG. 10B). ETP may therefore be a playerin several other tumor settings and may play a crucial role in cancercell behavior through both paracrine and autocrine signaling.

To identify the tissues that are critical targets for ETP incirculation, infrared fluorescent dye-labeled (IRD-800) recombinant ETPprotein was injected into PyMT mice through tail vein injection. Thesignal distribution in these tumor-bearing mice was compared with thatof WT mice. The in vivo fate of the labeled ETP was monitored byfluorescence scanning. A high fluorescence signal was observed in liverand bladder of all mice due to clearance. However, ETP was predominantlyobserved in tumor lesions compared with control-labeled IgGs (FIG. 2C).As determined by quantification, ETP was highly enriched in mammarytumor tissues relative to mammary glands of WT mice (FIG. 2D).

Elevated Local ETP Levels Convey Higher Antiapoptotic and PromitoticIndices in Normal Mammary Epithelial Cells.

To directly examine the role of ETP in mammary tumor growth, again-of-function approach was used with a transgenic mouse modelexpressing ETP under the control of MMTV promoter to elevate local ETPlevels within the mammary gland. To achieve efficient ETP secretion, aprolactin signal sequence was inserted in-frame 5′ to the regionencoding the mouse ETP sequence (FIG. 11A). ETP transgene levels werehighly upregulated in a high-expressing line compared with the moremodest overproduction of other low-expressing lines (FIG. 11B).Immunostaining with antibodies against ETP indicated that ETP wasenriched in mammary ductal epithelium in both transgenic mouse linesrelative to WT (FIG. 3A).

Assessment of mammary gland development in ETP mice is critical toevaluate the roles of ETP in mammary tumor progression, as most primarymammary tumors originate from mammary ductal or intraductal epithelialcells. Histological analysis—including whole-mount, H&E, and Masson'sTrichrome C staining—of mammary glands showed that ductal epithelialgrowth and the degree of fibrosis in both ETP transgenic lines wascomparable to those in WT mice (FIGS. 11C-11E). The final stages ofmammary gland development are completed upon pregnancy, lactation, andinvolution. The high-expressing ETP line displayed a deficiency infertility (FIG. 11F) and reduced locomotion (data not shown); thesesecondary effects of elevated ETP levels may make the interpretation oflocal findings within the mammary gland more challenging. The focus wastherefore placed on the low-expressing line and examined the process ofinvolution. In this process, the secretory epithelial cells undergoapoptosis with concomitant redifferentiation of adipocytes, therebyreconstituting prepregnancy status after weaning. Involution was delayedat an early stage of the process in ETP mice (FIG. 11G), along with areduction in apoptosis in secretory epithelial cells (FIGS. 3B and 3C).This suggests that ETP acts as a potent antiapoptotic factor in thissetting. Delays in the process of involution frequently resemble theprolonged survival of epithelial cells in a cancer setting.Nevertheless, ETP mice did eventually revert back to prepregnancystatus, albeit with delayed kinetics (FIG. 11G).

Abnormal developmental cues can induce and promote a canceroustransformation of mammary epithelial cells. Indeed, several mice withhigh ETP expression spontaneously developed tumors (FIG. 3D).Spontaneous tumor formation in low ETP expressers or WT mice up to 18months of age was not observed (data not shown). Immunostaining withKi67 showed that cell proliferation was increased in hyperplasticlesions of mammary tissue in the high-expressing mice (FIG. 3E), whichsuggests that high levels of ETP alone are sufficient to augmentpromitogenic activity. No lesions were observed in WT mice, and hence nodetectable mitogenic activity or Ki67 signal was evident (FIG. 3E).Further efforts were directed toward the physiologically more relevantlower-expressing ETP line, since these mice develop the mammary ductalepithelium completely normally and overexpress ETP only locally.

ETP Augments Tumor Growth and Metastasis in PyMT Mice.

To assess ETP function, the aim was to expose ETP mice to an additionaltumorigenic trigger. For this purpose, the PyMT mouse was used, anaggressive mammary adenocarcinoma model that develops late-stagecarcinoma and pulmonary metastasis within 15 weeks. Accumulated ETPlevels in tumor tissues of PyMT/ETP mice were about 1.5-fold those ofendogenous ETP in PyMT mice (FIG. 4A), and the rate of early tumorgrowth was augmented in PyMT/ETP versus PyMT mice (FIG. 4B). In light ofhigh endogenous ETP levels accumulating locally as well, differences inlate-stage tumors were not significant between the 2 groups when assayedby caliper measurements (FIG. 4C). More striking differences wereobtained at the level of metastasis (FIGS. 4D and 4E). To moresensitively assess tumor growth, a mouse model was generated harboringthe infrared fluorescence transgene FP635 under the control of the MMTVpromoter, which allowed monitoring of the tumor growth longitudinallythrough in vivo imaging (FIG. 12A). Breeding this transgene into PyMTanimals (referred to herein as FP635/PyMT mice) allowed readilyassessing of the tumor growth by whole-body fluorescence signalintensity, given that the infrared range allows deeper tissuepenetration with reduced autofluorescence from surrounding AT (FIG.12B). By assessing in vivo images, the differences of tumor burdenbetween the FP635/PyMT/ETP and FP635/PyMT groups became apparent andsignificant (FIG. 4F), which indicates that ETP not only promotespulmonary metastasis, but also further enhances primary tumor growth.Ki67 staining at late stages did not show an increased frequency ofproliferating cells in tumor tissues in PyMT/ETP versus PyMT animals(FIG. 5A). Nevertheless, tumor tissue fibrosis at that age was doubledin PyMT/ETP versus PyMT mice (FIG. 5B). Indeed, a subset of genesassociated with tissue fibrosis, including several types of collagens,lysyl oxidase (Lox), and TGFβ, as well as genes forepithelial-mesenchymal transition (EMT), such as fibroblast stimulatingprotein (FSP1) and vimentin, showed trends toward an increase in tumortissues (FIGS. 5E and 5F). More dramatic alterations were obtained inthe area of tumor angiogenesis: ETP tumor tissues harbored a 3-foldincrease in functional blood vessel area compared with controls (FIG.5C), with a concomitant reduction in hypoxia (FIG. 5D). Markers forangiogenesis, such as CD31, VEGFR2, and HIF1α, were significantlyupregulated in tumor tissues from PyMT/ETP mice. Moderate increases wereobserved in lymphangiogenesis markers, such as VEGFc, podoplanin (Pdpn),and the lymphatic vessel endothelial hyaluronan receptor (LYVE-1) (FIG.5G). These gene expression changes were consistent with the immunostainsof tumor tissues for lymphangiogenesis, such as podoplanin (FIG. 5C).Levels of inflammatory cytokines, such as IL6 and TNFα, were moderatelyincreased (FIG. 5H). Taken together, these findings indicate that ETPenhances fibrosis, angiogenesis, and inflammation, all of which canpromote primary tumor growth and metastasis.

ETP Enhances the EMT Process.

To investigate gene expression alterations induced by ETP, cDNAmicroarrays were compared from size-adjusted tumor tissues from PyMT/ETPand PyMT mice. ETP-modulated genes fell primarily into categories oftargets involved in key phosphorylation events, such as phosphatases,kinases, and other phosphoproteins (FIG. 13A). Furthermore, the mostsignificantly altered canonical pathways modulated by ETP wereassociated with immune responses, cell cycle regulation, and stem cellpluripotency (FIG. 13B). Notably, stem cell-like pluripotency is ahallmark of cancer cells for survival and invasion, which is tightlylinked to the process of EMT. Levels of the epithelial cell markerE-cadherin—the loss of which is a characteristic feature of EMT—weresignificantly decreased in tumor tissues from PyMT/ETP relative to PyMTmice (FIG. 6A), which suggests that ETP induces EMT. This enhanced EMTwas consistent with the in vivo phenotype: the metastatic burden wasprominently increased in PyMT/ETP versus PyMT mice (FIGS. 4D-4F).

Effects of ETP Synergize with the Canonical TGF-β Pathway to PromoteLung Metastasis.

To delineate the mechanism underlying the increase in EMT processes inPyMT/ETP mice, the effects of ETP on the TGF-β pathway wereinvestigated. TGF-β signaling has previously been implicated inEMT-associated tumor growth and metastasis, which are associated withthe acquisition of metastatic traits. To examine whether ETP signalingconverges with the canonical TGF-β pathway, 2 ETP constructs weregenerated: a form that was secretion incompetent, and thus retainedwithin the secretory pathway, as well as a secreted form (FIG. 14A).These constructs were used for a reporter assay with TGF-β-dependentSmad protein—binding elements (SBEs). Interestingly, only the secretedform of ETP enhanced SBE reporter activity; furthermore, this wascritically dependent on TGF-β stimulation (FIG. 6B), which indicatesthat ETP synergizes with the TGF-β pathway through cell-surfaceinteractions. This is based on the fact that the enhanced TGF-βsignaling synergistically activated through ETP was completely abolishedby treatment with the monoclonal TGF-β neutralizing antibody 1D11 (FIG.6C), which strongly suggests that ETP-dependent SBE reporter activationand consequent signaling events fully rely on the presence of TGF-β.

To further elucidate the TGF-β-dependent role of ETP as a tumorenhancer, in a less aggressive experimental setting of tumorprogression, an allograft model was used in which TGF-β signaling wasinhibited with TGF-β neutralizing antibodies in the context of ETPoverexpression. Although tumor growth for Met-1 cells was significantlyenhanced by ETP, TGF-β inhibition did not efficiently reduce ETP-inducedtumor growth (FIG. 6D), which suggests that TGF-β-mediated signaling isless relevant for the growth of primary tumors in the ETP-expressingtumor stroma. Histological analysis of tumor tissues revealed anincrease in mesenchymal-like stromal cells in the ETP-expressing tumorstroma, a phenomenon that was reversed by TGF-β inhibition (FIG. 6E).This further suggests that the TGF-β pathway participates in ETP-inducedEMT. Additionally, the ETP-mediated increase in tissue fibrosis wasattenuated by TGF-β inhibition (FIG. 6F). These results weresubsequently confirmed by examining EMT markers in tumor tissues (FIG.6G), namely E-cadherin, vimentin, and α-SMA, which is an activatedmyofibroblast marker widely used for EMT assessment. Of note,manipulation of TGF-β signals in tumor tissues using genetic mousemodels for TGF-β, TGF-βR1, and TGF-βRII have highlighted that TGF-βsignaling augments cancer cell invasiveness, primarily throughstimulation of EMT processes, enhancing metastatic rather than primarytumor growth. The efficacy of TGF-β neutralizing antibodies on themetastatic potential of cancer cells from PyMT/ETP and PyMT mice(referred to herein as ETP⁺- and Ctrl-cancer cells, respectively) wassubsequently investigated. ETP⁺-cancer cells metastasized at a higherrate than Ctrl-cancer cells (FIG. 6H). Moreover, this increased rate ofmetastasis was attenuated by TGF-β inhibition (FIG. 6H). Collectively,these results suggest that the TGF-β-dependent aspects of ETP actionrelate only to the acquisition of cancer cell invasive and metastatictraits, not to primary tumor growth.

ETP, as a Potent Chemokine, Augments Primary Tumor Growth ThroughTumor-Stromal Interactions.

To examine cancer cell-autonomous effects, allografts were performedwithout Matrigel plugs. Tumor growth of ETP⁺-cancer cells was comparableto Ctrl-cancer cells (FIG. 15A). Indeed, in vitro examination of mammarycancer cells, including Met-1 and MCF-7 cells, revealed that cellproliferation was not affected by addition of purified ETP (FIGS. 15Band 15C). However, the vascularization of allografts of ETP⁺-cancercells was significantly increased (FIG. 15D). This suggests that cancercells per se are not responsive to ETP with respect to growth, but thatETP augments endothelium formation; therefore, tumor stromal, ratherthan cancer cell—autonomous, interactions account for the increase intumor growth observed in vivo. To test this, ETP⁺- and Ctrl-tumors wereimplanted into isogenic WT mice. ETP⁺-tumors grew dramatically fasterthan did Ctrl-tumors (FIG. 7A). From these allograft studies, the potentproangiogenic, profibrotic, and proinflammatory effects of ETP,initially observed in the PyMT setting (FIG. 5), became evident (FIG.16). In light of this, stromal effects on primary tumor growth were thefocus. These stromal effects were mediated by the tumor-associatedvasculature, as well as fibrotic and inflammatory pathways. Majorstromal target cell types involved in tumor interactions wereendothelial cells, fibroblasts, and macrophages, all of which haveestablished roles in tumor growth and metastasis.

In light of the findings above, it follows that ETP may function as achemokine during tumor stroma expansion, recruiting or possiblyactivating stromal cells to support tumor growth. In vivo targeted cellrecruitment studies revealed that Matrigel plugs combined withrecombinant ETP and injected into mammary fat pads of WT mice recruitedsignificantly more stromal cells than did PBS (FIG. 7B). Monoclonalanti-ETP antibodies were subsequently generated to effectivelyneutralize ETP, thereby generating an ETP-based therapeutic approach(FIG. 9C). Chemokine activity of ETP was completely blocked by 10B6, butnot by 1D11 (FIG. 7B), which suggests that ETP-inherent chemokineactivity was independent of TGF-β signaling. Furthermore, the majorityof cells recruited into the Matrigel plugs by ETP were CD31⁺ endothelialcells (FIG. 17A). More specifically, mammary epithelial cancer cellswere cocultured with either mouse endothelial cells (MS-1; FIGS. 7D-7E)or primary macrophages (FIGS. 7F-7G) in a Transwell plate (FIG. 7C), andthe migration of the MS-1 cells and macrophages were subsequentlyquantified (FIG. 7H). ETP⁺-cancer cells recruited substantially moreendothelial cells and macrophages (FIGS. 7E, 7G, and 7H). This suggeststhat the majority of chemoattractant properties exerted by COLE areexerted by ETP.

In vitro cell migration assays revealed that ETP recruited twice as manyMS-1 cells than did controls (FIG. 7I). Furthermore, in vitroangiogenesis assays using MS-1 cells demonstrated that endothelial cellsincubated with conditioned media from ETP-overexpressing HEK-293T cellsmobilized and organized vasculature structures much more actively thanconditioned medium harvested from control HEK-293T cells (FIG. 17B).10B6 completely blocked these effects (FIG. 17B). Thus, ETP plays acrucial role in endothelial cell recruitment, migration, and vesselformation during the process of angiogenesis. Based on theseobservations, it is concluded that ETP is critical for the recruitmentof stromal cells into the tumor microenvironment through its action as achemokine.

A Neutralizing Anti-ETP Monoclonal Antibody Attenuates Tumor Growth byInhibiting ETP-Mediated Expansion of the Tumor Stroma.

It was next examined whether ETP neutralization can attenuate tumorprogression. Of note, PyMT mice expressed high levels of endogenous ETPin the tumor-infiltrated stromal compartment (FIG. 1E). Tumor growth wassignificantly attenuated by 10B6 treatment compared with isotype controlIgG treatment (FIG. 8A). Similarly, FP635/ETP⁺-cancer cells wereimplanted into WT mice, with or without 10B6 added to the Matrigel plug;tumor growth was then monitored using fluorescence imaging. The rate ofETP⁺-cancer cell growth was significantly higher relative to Ctrl-cancercells; however, their growth was effectively attenuated by 10B6 (FIG.8B). Histological analysis of tumor tissues indicated that variousstromal cells had the capacity to infiltrate into the Matrigel plugscontaining ETP⁺-cancer cells. Again, 10B6 inhibited ETP-mediated tumorstromal expansion and eventually triggered tumor regression (FIG. 8C).ETP⁺-cancer cell allografts consistently displayed higher levels offibrosis (FIG. 8D) and a high degree of stromal cell infiltration,including by α-SMA⁺ cells, FSP-1⁺ fibroblasts, and CD31⁺ endothelialcells in addition to F4/80⁺ macrophages (FIGS. 8E-8H). These cells werehighly proliferative, as demonstrated by Ki67⁺ staining (FIG. 8I). Asexpected, the ETP-mediated increase in fibrosis and stromal expansionwas completely blocked by 10B6 (FIG. 8D-8H). Collectively, the resultsindicated that stromal adipocytes play a crucial role in mammary tumorprogression; that ETP is a powerful stromal factor that exerts a majorinfluence on primary tumors through its chemokine activities; and thatETP can affect metastatic growth via TGF-β-mediated EMT. Thus,ETP-directed approaches may serve as novel therapeutic regimens in thetreatment of breast cancer (FIG. 8J).

Discussion

A prominent environmental stimulus of tumor dissemination is hypoxia,triggered by a high demand for cell proliferation and insufficientangiogenesis. Comparable to this process, hypertrophic AT expansionduring obesity can also trigger local hypoxia that can further progressto AT fibrosis. These obesity-related pathophysiological changes canlead to an environment that is conducive to cancer growth, such aschronic inflammation, inadequate angiogenesis, and enhanced fibrosis. Inthis setting, obesity may contribute to an ETP-rich tumormicroenvironment through a positive feed-forward mechanism. Indeed,COL6α3 message levels are upregulated in obese AT. COL6 upregulation hasbeen reported in various aspects of tumor progression. Malignant cancercells can also express COL6; this has been reported for the mammarygland, the colon, pancreatic ductal adenocarcinomas, andhepatocarcinomas. Thus, the source of ETP in the tumor microenvironmentmay be heterogeneous, with signals cooperatively influencing cancer cellbehavior through paracrine and autocrine pathways. Nevertheless, stromaladipocytes represent a prominent source for COL6 in the mammary tumormicroenvironment (FIG. 18A). However, the detailed mechanisms underlyingthe specific effects of COL6 on tumor behavior have not previously beenelucidated. Here, the above results show that ETP, a cleaved product ofCOL6, can be a critical mediator of several tumor-associated phenomenaand is of particular importance in tumor progression in the context ofobesity.

Within the tumor milieu, EMT is initiated by extracellular stimuli. Thiscan be exerted by ECM components (collagens, fibronectin, hyaluronicacids, and MMPs) as well as by certain growth factors (TGF-β, EGF, andHGF), all of which are provided by both paracrine and autocrine signalswithin the tumor microenvironment. One of the prominent ECM moleculesreleased from stromal adipocytes is COL6. As a COL6 processing product,ETP plays an important role in the local microenvironment, stimulatingTGF-β-dependent EMT in the context of mammary tumors to potentiateprometastatic effects (FIG. 6). Gene expression profiling andimmunostaining of tumor tissues from PyMT/ETP mice confirmed enhancedETP-mediated acquisition of EMT characteristics, whereas in vitro dataindicated that ETP alone did not induce EMT (data not shown). Thissuggests that ETP may function as an important costimulator of existingpathways for the EMT, such as TGF-β signaling and possibly activation ofintegrins and Wnt signals.

Increased tissue fibrosis, combined with high tissue rigidity (due toECM remodeling and crosslinking), is positively associated with tumorgrowth. The results above revealed an ETP-induced fibrotic environment,with high levels of myofibroblast accumulation within tumor tissues, asa key characteristic of ETP action. These activated myofibroblasts inETP⁺-tumors were derived, at least in part, by EMT. In addition, ETP mayfacilitate additional processes, such as microfibril assembly ofpreexisting collagen fibers or stimulation of myofibroblastdifferentiation. Moreover, promoting transformed mesenchymal cellproliferation can enhance the appearance of additional stromal cells;ETP may also effectively promote this process. Indeed, blocking the EMTby using a TGF-β neutralizing antibody did not completely eliminatefibrosis in ETP⁺-tumors (FIG. 6F). These data indicate that theETP-induced EMT and subsequent fibrotic traits in tumors contribute toan increase in tumor growth and metastasis, which highlights a centralrole for ETP in tumor progression.

Evidence was also provided herein for the potent ETP-mediatedchemoattractant properties. These ETP effects can even be mimicked in atumor-free environment. A number of reports highlight significantcorrelations between COL6α3 and chronic inflammation, based on increasedmacrophage infiltration into AT depots of obese subjects. TheETP-mediated chemoattractant properties described herein may offer amechanistic basis for these clinical correlations. Neutralizing theseETP-mediated effects in normal, tumor-free AT may yield beneficialoutcomes as well. The current efforts are directed towardadipocyte-derived overexpression of ETP, to examiner whether a localexcess of ETP will exert beneficial effects (due to its proangiogenicproperties) or negative effects (due to its proinflammatory andprofibrotic properties) on a fat pad not challenged with an invadingtumor.

Fibrosis in obese AT is associated with an increase of various MMPs orTIMPs resulting in collagen degradation. MMP-11, MMP-2, and MMP-9 havebeen suggested as peptidase for COLE, although there is no furtherevidence whether these MMPs cleave ETP. Based on the fact that mostcancer cells express high levels of MMPs associated with tumor growthand metastasis, it is likely that there are abundant sources for ETPcleavage activity within the tumor microenvironment. The identificationof the critical protease involved in ETP processing may offer a newapproach to curbing growth by pharmacologically inhibiting this step.The findings herein unveiled an important role of the adipocyte as anactive component of the tumor stroma that actively interacts with cancercells and a number of other relevant local cell types. The data hereinalso highlights that an adipocyte-derived ECM cleavage product activelycontributed toward the remodeling of the tumor microenvironment byenabling the progression of tumor growth and metastasis throughenhancement of the EMT process and subsequent chemotaxis of endothelialcells and macrophages. In many aspects, the deposition of ECMcomponents, such as ETP in the tumor stroma, resembles a wound-healingprocess, as this involves the recruitment and stimulation of immunecells, endothelial cells, and fibroblasts during the wound repairprocess. However, unlike during the wound-healing process, ETP promptscancer cells to sustain mesenchymal cell-like traits and activatesfibroblasts in the tumor stroma, drastically increasing local fibrosisand eventually enhancing metastatic growth. The findings herein havefurther implications for several tissues that have an associatedpathological fibrotic component, such as the liver, cartilage, lung, andheart; COLE expression has been documented in all these tissues. Furtherefforts targeted toward ETP neutralization in various pathologicalsettings can establish this approach is a viable antifibrotic strategythat is generally beneficial, not only in the setting of tumorprogression and metastasis, but also during normal AT expansion.

Methods

Mice.

See Supplemental Methods for detailed information on the mice usedherein. All experiments were conducted using littermate-controlledfemale mice. All animals used in this study were in a pure FVBbackground.

Tumor Imaging.

FP635/PyMT mice or cancer cells isolated from tumors from FP635/PyMTmice were imaged with an IVIS scanner (Caliper Lifesciences), and thesignal intensity was analyzed with Living Image version 3.2 (CaliperLifesciences). See Supplemental Methods for conventional analyses oftumor growth and metastasis.

ETP-Specific Polyclonal and Monoclonal Antibodies.

ETP-GST fusion proteins for both mouse and human ETPs were purified frombacteria and used as antigens for polyclonal antibodies (Covance). Formonoclonal antibody generation, native ETP was purified by gelfiltration chromatography (GE Healthcare) from conditioned media of amouse ETP-overexpressing HEK-293 stable cell line. See FIG. 1A for ETPamino acid sequences.

Quantitative RT-PCR.

Total RNA was isolated using the RNeasy kit (Qiagen) following tissuehomogenization in TRIzoL (Invitrogen). Total RNA (1 μg) was reversetranscribed with SuperScript III reverse transcriptase (Invitrogen).Quantitative real-time PCR (qRT-PCR) was performed using RocheLightcycler 480. Primer sequences used in this study have been describedpreviously.

Analysis of ETP Homing.

The homing of ETP in circulation was determined by injectingfluorescently labeled ETP into tail veins. ETP and IgGs were labeledwith IRDye800 CW NHS Ester (Licor Bioscience) at a 1:1 molar ratio(dye/protein), according to the manufacturer's instructions. Whole-bodyfluorescence images were collected on the Odyssey scanner (LicorBioscience). All scans were performed under anesthesia (Aerrane) usingan EZ-2000 Microflex small-animal anesthesia system (EZ Systems). At theend of experiments, each organ was collected and imaged for signalintensity with the Odyssey scanner. Quantified values were normalized tothe total area of each organ.

Histological Analysis.

Formalin-fixed paraffin-embedded tissue sections were used forimmunohistochemistry. Deparaffinized tissue slides were stained with theprimary antibodies shown in Supplemental Methods. Staining forfunctional blood vessels and hypoxic lesions as well as whole-mountstaining of mammary glands were followed as described previously. TUNELassays were performed according to the manufacturer's protocol (TrevigenInc.). Masson's Trichrome C and H&E staining were performed by J.Shelton (University of Texas Southwestern Medical Center, Dallas, Tex.,USA). Deidentified human tumor samples were obtained from the Universityof Texas Southwestern Tissue Resource.

Primary Culture of Mammary Cancer Cells and Implantation.

Isolation of mammary epithelial cancer cells and implantation procedureswere as previously described. Tumor growth was monitored once weeklybeginning 2 weeks after implantation.

In Vitro Cell Migration Assay.

Thioglycollate-elicited macrophages or MS-1 in serum-free media wereloaded into the upper chamber of a Transwell plate (8 μm pore size;Costar). As chemoattractants, ETP or the indicated cancer cells wereadded to the bottom chamber with DMEM containing 2% FBS. 18 hours later,cells on the underside of the membrane were fixed with 10% formalin,stained with hematoxylin, and counted. Images were acquired using theNikon Cool Scope (Nikon).

Statistics.

Data are presented as mean±SEM. Data were analyzed by 2-way ANOVAfollowed by Newman-Keuls multiple comparison test or by 2-tailedStudent's t test, as appropriate, with GraphPad Prism version 5software. A P value less than 0.05 was considered statisticallysignificant.

Study Approval.

This study was carried out in strict accordance with the recommendationsof the NIH Guide for the Care and Use of Laboratory Animals. All animalexperiments were approved by the Institutional Animal Care and ResearchAdvisory Committee at the University of Texas Southwestern MedicalCenter (protocol no. 2010-0006). All efforts were made to minimizeanimal suffering.

Supplemental Methods

Mice.—

Endotrophin transgenic mice were generated by fusing cDNA encoding themouse COL6A3-C5 domain (amino acids 2590-2657, NP_034056) to theprolactin signal sequence at the amino-terminus(ATGGACAGCAAAGGTTCGTCGCAGAAAGGGTCCCGCCTGCTCCTGCTGCTGG TGGTGTCAAATCTACTCTTGTGCCAGGGTGTGGTCTCC; SEQ ID NO: 14), which allows endotrophin to besecreted from the cells, into a plasmid containing the 3.2-kb MMTV(mouse mammary tumor virus) promoter and 3′-SV40 region.Transgene-positive offspring were genotyped using PCR with the followingprimer set: 5′-ACGAGAACAGATTCCACTCC-3′ (SEQ ID NO: 15) and5′-TCAGCAGTAGCCTCATCATCAC-3′ (SEQ ID NO: 16). Infrared fluorescentprotein FP635 transgenic mice were generated by subcloning the FP635domain originated from plasmid pTurboFP635 (Evrogen) into a plasmidcontaining the 3.2-Kb MMTV promoter and a conventional 3′-UTR region.Genotyping was performed using PCR with following primer set:5′-AGAGACCTACGTCGAGCAGC-3′ (SEQ ID NO: 17) and5′-GGGTCCATGGTGATACAAGG-3′ (SEQ ID NO: 18).

Reagents.

Primary antibodies used in histological analysis: rabbit polyclonalagainst holo-COL6 (Abcam, Ab6588), CD31 (Abcam, ab38364), α-smoothmuscle actin (Abcam, ab5694), FSP1 (Abcam, ab27957), Vimentin (CellSignaling, #5741) and E-cadherin (Cell Signaling, #3195); mousemonoclonal F4/80 (invitrogen, MF48000) and cytokeratin (Cell Signaling,#4545); rat monoclonal Ki67 (Dako Cytomation) and endomucin (Santa Cruz,sc-65495). TGFβ neutralizing antibody, 1D11 was generously providing byDr. Rolf Brekken (UTSW Medical Center, Dallas).

Analysis of Tumor Progression and Lung Metastasis.

Tumor onset was monitored twice weekly by palpation. Tumor sizes weremeasured with a digital caliper twice weekly and the volumes werecalculated as (length×width)/2. Inguinal tumors were weighted todetermine tumor burden. Animals were sacrificed when the tumor burdenvisibly affected the host or when the tumors reached the IACUCpredetermined limit of 20 mm along one axis. Metastatic tumor growth wasdetermined by histological analysis with H&E stained lung tissues.

Immunoblotting.

Cell lysates were harvested using NP-40 lysis buffer, supplemented withphenylmethylsulfonyl fluoride (PMSF, 1 mM), protease inhibitor (Roche)and phosphatase inhibitor (Roche). Protein samples were immunoblottedusing standard methods. For the culture media, differentiated 3T3-L1adipocytes and preadipocytes were serum starved in DMEM media. Followingovernight incubation, media was harvested and filtered (Millipore, 0.45mm). The conditioned media was concentrated using centrifugal filters(Amicon Ultra, 3K) at 14,000 g for 40 min. Secreted ETP was detectedusing α-mouse ETP polyclonal antibody compared to COLE (Abcam, Ab6588).The primary antibodies were detected with secondary IgG-labeled withinfrared dyes emitting at 700 and 800 nm and visualized on the LicorOdessey Infrared Scanner. The scanned results were analyzed using theOdessey v2.1 software (Licor Bioscience). The complete unedited blotsfor all Western blotting images in the main are shown in theSupplementary images.

Involution.

8-week-old females were housed individually upon pregnancy. Immediatelyafter birth of their littermates, litter sizes were standardized to 6pups per mother in order to prevent inter-mouse mammary gland variation.Involution was initiated by the removal of pups after 10 days ofsuckling. Mammary glands were collected for fixation at 0, 2, 3, 5 daysafter forced weaning and stained with H&E.

Assessment of Reproduction.

8-week-old female mice (5-9 mice/group) were mated with wild type males.Each female was monitored for resulting pregnancies. Litters weremonitored for survival to weaning age.

Microarrays.

Total RNA was extracted from tumor tissue from 12-week-old PyMT andPyMT/endotrophin (n=12/group). Microarray experiments were performed bythe UTSW microarray core facility. The Mouse Illumina Bead Arrayplatform (47K array) (Illumina, Inc.) was used in this study. Gene listsand cluster analyses of the data sets were performed using IngenuityPathway Software (Ingenuity systems) and David Bioinformatics Resource(http://david.abcc.ncifcrf.gov/). Gene profiling data are available fromGEO (http://www.ncbi.nlm.nih.gov/geo) under accession no. GSE39622.

In Vitro Angiogenesis.

A total 300 μl/well of growth factor reduced matrigel (BD biosciences)was plated into 12 well plates. MS-1 cells (5×10⁴) were seeded andimages were acquired 3-4 hours after incubation with the conditionedmedia indicated.

Luciferase Reporter Assay.

Cell lysates were harvested and analyzed for luciferase reporter assaysfollowing the manufacturer's protocol (Applied Biosystems, TheDual-Light luminescent reporter gene assay). The pGL3-SBE reporter,pGL3-βGal and indicated ETP constructs; pRA-ctrl (empty vector),pRA-sETP (secretion form of ETP) and pRA-ETP (intracellular form of ETP)were transiently transfected into Met-1 cells. 1 day after transfection,TGFβ (5 ng/ml) with either 1D11 (5 μg/ml) or IgG (5 μg/ml) was addedovernight. Total cell lysates were analyzed for luciferase activity.

Example 2: Inhibition of Endotrophin, a Cleavage Product of Collagen VI,Confers Cisplatin Sensitivity to Tumors

The therapeutic benefit of cisplatin in human cancer treatments is oftenlimited due to resistance. Thiazolidinediones (TZDs) (peroxisomeproliferator activated receptor γ agonists) show beneficial effects inthe context of cisplatin-based chemotherapy. Previous work indicatesthat collagenVIα3 (COL6A3) plays an important role in cisplatinresistance. However, the detailed molecular mechanisms underlying thecorrelations between COL6A3 and cisplatin resistance remained elusiveprior to this study. The goal of this study was to elucidate the rolesof endotrophin, a cleavage product of COL6A3, in cisplatin resistanceand elaborate further to see if endotrophin modulates the beneficialeffects of TZDs in cisplatin therapeutics in breast cancer.

Endotrophin, which is mainly secreted from stromal adipocytes in thetumor microenvironment, was demonstrated herein to confer a high degreeof cisplatin resistance by enhancing epithelial-mesenchymal transition,fibrosis and angiogenesis. Furthermore, the powerful beneficial effectsof TZDs on cisplatin sensitivity are mainly due to a marked inhibitionof endotrophin-mediated activities. This suggests that TZDs directlymediate enhanced cisplatin chemosensitivity through a downregulation ofendotrophin. Treatment with an endotrophin neutralizing monoclonalantibody in combination with cisplatin very effectively inhibits tumorgrowth of allografts of MMTV-PyMT tumors.

It is well appreciated that chemo-responsiveness is changed over thecourse of tumor progression, and it varies greatly between differenttumor types; identifying the critical players mediating thischemo-resistance is important to devise better therapeutic strategies.The results herein have important clinical implications, as endotrophinis increased in tumors upon chemotherapy, and the associated EMT is apredictor of chemo-resistance. Therefore, endotrophin levels can be astrong prognostic marker with respect to the tumor response tocombination therapy of TZDs with cisplatin, and the neutralization ofendotrophin can further improve the therapeutic response to combinationtherapy.

Cisplatin Augments COL6A3 Levels, Whereas TZDs Cause a Reduction.

To assess the beneficial effects of TZD (using mostly the TZDrosiglitazone) on platinum-based chemotherapies in mammary tumor modelsin vivo, either a MMTV-PyMT (“PyMT”) mouse model or an allograft ofMet-1 cancer cells (originating from MMTV-PyMT mammary tumors) that wastransplanted into isogenic wild-type mice were used. To visualizesystem-wide tumor burden in vivo, an infrared-fluorescent protein(FP635) overexpressing transgene driven by the MMTV promoter(MMTV-FP635) was introduced into PyMT mice (Park & Scherer, 2012b).Tumor regression was monitored by utilizing fluorescence scanning overthe course of cisplatin treatment (FIG. 19A). Tumor growth wasefficiently reduced and pulmonary metastasis were also slightlyattenuated in PyMT mice exposed to TZDs (20 mg/kg) in combination withcisplatin (1 mg/kg) compared to those mice given only cisplatin (FIG.19A). Met-1 allografts showed a better response to the combination ofTZD with cisplatin than the response seen in PyMT mice (FIG. 19B). Thismay be due to PPARγ-dependent activation of intrinsic oncogenicpathways, such as wnt, or contributions of the tumor stroma respondingto a prolonged treatment of TZDs, which may counteract their beneficialeffects on cisplatin in the PyMT mice (Saez et al, 2004). In addition,it was previously shown that TZDs are potent inducers of the adipokineadiponectin that were implicated in enhanced angiogenesis and improvedcellular survival (Landskroner-Eiger et al, 2009). Subsequenthistological analysis of tumor tissues indicated that cancer cell deathwas increased about 2-fold with the TZD combination (FIG. 23A). The factthat the metallothionein (MT) levels, a molecular marker for drugresistance (Theocharis et al, 2003), are suppressed by the TZDcombination with cisplatin, is well appreciated (Girnun et al, 2007).Consistently, immunostaining for MT in tumor tissues of PyMT mice showedthat cisplatin treatment significantly increased the MT levels, and thiswas suppressed in the presence of TZD (FIG. 23B). As such, the PyMT miceserve as a useful model to assess the beneficial effects of TZDs inplatinum-based therapeutics in vivo.

To see whether COL6 is involved in the beneficial effects of TZDs onplatinum-based therapy, the expression levels were determined for COL6in response to chemotherapy. The mRNA levels of COL6A3 in tumor tissuesof PyMT mice were significantly increased in response to cisplatintreatment; this increase was dramatically suppressed by combination withTZDs (FIG. 19C). These results indicate that COL6A3 levels may have animpact on the degree of chemo-sensitivity between TZDs and platinum invivo.

Cisplatin Augments Epithelial-Mesenchymal Transition, Whereas TZDAttenuates it.

The EMT process in tumor tissues is well known to contribute to anacquired drug resistance (Arumugam et al, 2009; Latifi et al, 2011).This suggests a fundamentally reduced sensitivity of mesenchymal-likecells to chemotherapeutic approaches. Targeting the critical factorsthat contribute to the EMT process, such as Snail, Slug and Twist1, hasbeneficial effects for cisplatin-based therapies (Haslehurst et al,2012; Zhu et al, 2012), further generalizing a model that correlates thedegree of cisplatin sensitivity with the EMT status of tumor tissues.Moreover, TZDs have been suggested to suppress EMT, resulting in areduced level of tumor metastasis (Reka et al, 2010). In the mousemodels, the mRNA levels for transcription factors associated with EMT,such as Snail, Slug and Twist1, were significantly increased in responseto cisplatin exposure. The increases in critical mediators of EMT,especially the increased levels of Twist1, were significantly attenuatedby combination treatment with TZD (FIG. 19D). This supports the ideathat cisplatin induces EMT in cancer cells, and that the beneficialeffects of TZDs in the context of cisplatin exposure are partly mediatedby suppression of EMT. This is substantiated by immunohistochemistrywith critical EMT markers. Immunostaining with antibodies against EMTmarkers that include either the loss of E-cadherin or an increase invimentin expression, showed a significant increase of EMT followingcisplatin treatment in tumor tissues, whereas levels of E-Cadherin weresustained in TZD and cisplatin treated group (TZD/CIS) comparable tocontrol tissues (ND/PBS) (FIG. 19E). In parallel, cisplatin-inducedincreases in vimentin levels were also significantly reduced bycombination with TZD (FIG. 19F). These observations led to studies totest whether endotrophin plays a critical role in the cisplatin-drivenincrease of EMT, as endotrophin plays a generalized role in EMT in tumortissues (Park & Scherer, 2012b). It was examined whether theTZD-mediated decrease in COL6A3 levels (FIG. 19C) can be connected tothe TZD-mediated enhanced cisplatin sensitivity through suppression ofthe endotrophin-induced EMT.

Endotrophin, a Cleavage Product of COL6A3, Confers Cisplatin Resistancein Tumor Tissues.

It has been previously shown that MMTV-Endotrophin mice bred to the PyMTmice (PyMT/endotrophin) develop more aggressive tumors compared to PyMTmice (Park & Scherer, 2012b). Here, these mice were further examined tosee whether endotrophin induces cisplatin resistance. PyMT/endotrophintransgenic mice were treated with either PBS or cisplatin and comparedto PyMT control littermates. Primary tumor growth of PyMT mice waseffectively curbed with a high dosage of cisplatin treatment (2.5 mg/kg,ip. twice a week), whereas PyMT/endotrophin mice were resistant to theeffects of cisplatin treatment (FIG. 20A). Similarly, allografts oftumor pieces taken from PyMT and PyMT/endotrophin mice transplanted intoisogenic wild-type mice showed that endotrophin⁺-tumors were moreresistant to a lower dosage of cisplatin treatment (1 mg/kg, ip. twice aweek) compared to control (Ctrl)-tumors (FIG. 20B-20C). These resultsfurther corroborate a direct connection of cisplatin-induced high levelsof endotrophin expression and chemo-resistance.

Acquisition of the Beneficial Effects of TZDs to Cisplatin CriticallyDepends on the Endotrophin Levels

It has been shown above that the beneficial effects of TZDs on thecisplatin therapeutic efficiency are linked to endotrophindown-regulation. Do the TZD effects converge on to theendotrophin-mediated signaling pathways? Both mRNA and protein levelsfor endotrophin were dramatically reduced with the TZD and cisplatincombination treatment. Therefore, it was assessed whether endotrophinoverexpression could abolish the beneficial effects of TZD on cisplatinefficacy. Endotrophin⁺-cancer cells originating from PyMT/endotrophinmice were compared to Ctrl-cancer cells from PyMT mice, and wereimplanted into wild-type mice. TZD was given to wild-type hosts 10 daysprior to implantation and cisplatin was injected intraperitoneally every5 days, starting 3-weeks post implantation when the tumor volume reached100 mm³ (FIG. 21A). Endotrophin⁺-tumors were more resistant to cisplatintreatment compared to Ctrl-tumors (FIG. 21B, Ctrl/ND vs. ETP/ND), andthis increase was markedly attenuated by the combination with TZD (FIG.21B, ETP/ND vs. ETP/TZD). This suggests that TZD influences not only theendotrophin expression per se, but it may also impact the downstreampathways of endotrophin. Endotrophin-independent pathways may be alsocontributing, or TZD may act on host endotrophin levels in thistransplantation paradigm. Defined necrotic lesion areas, as assessed byH&E stains, were significantly decreased in endotrophin⁺-tumors. Thisphenomenon was however reversed by combined treatment of cisplatin withTZD (FIG. 21C). This suggests that a combination of TZD with cisplatinconfers sensitivity to endotrophin⁺-tumors. Accordingly, the significantendotrophin-mediated increase on EMT, angiogenesis and fibrosis seen inendotrophin⁺-tumors was suppressed by the combination of cisplatin withTZD, as judged by immunostaining for vimentin (EMT), lectin perfusion(angiogenesis) and Masson's trichrome C stain (fibrosis), respectively(FIGS. 21D-21F). This suggests that TZD attenuates the downstreamsignaling pathways induced by endotrophin.

The Suppression of Endotrophin Activity can be Achieved by Either UsingTZD or Anti-Endotrophin Monoclonal Antibodies, Both of which SensitizeTumors to Cisplatin Therapeutics.

As a last step, therapeutic potential of a previously describedendotrophin neutralizing antibody (clone 10B6) on cisplatin sensitivitywas determined. Tumor pieces taken from PyMT mice were implanted intowild-type mice and treated with cisplatin alone or in combination witheither TZD or 10B6 once the tumor volume reached 100 mm³. Tumorregression was monitored for 2-months post implantation. Both TZD and10B6 treatment are demonstrated to efficiently sensitize the tumors tocisplatin treatment (FIG. 22A). Xenograft models were utilized with themammary carcinoma cell line 4T1, which is highly invasive and rapidlymetastasizes throughout the body, resembling human stage IV breastcancer (Pulaski & Ostrand-Rosenberg, 1998). Nude mice were injected with4T1 cells in mammary adipose tissues. Treatment with cisplatin wasinitiated when the tumor volume reached at 100 mm³. Treated was combinedwith either control IgGs or 10B6. Treatment of either 10B6 or cisplatinalone for 28-days barely inhibited primary tumor growth of 4T1, whiletreatment with cisplatin combined with 10B6 induced a moderate, butsignificant inhibition in comparison to cisplatin or 10B6 alone (FIG.22B). However, most prominent effects of combination treatment(cisplatin and 10B6) were observed on metastatic growth. The metastaticburden on the lung, as determined by assessing the metastatic lesionareas, was significantly attenuated by combination treatment relative toindividual treatments (FIG. 22C). Notably, the combination of cisplatinand endotrophin neutralization showed a particularly higher efficacy onmetastatic growth than either treatment alone for late stages of 4T1carcinomas. Furthermore, a subset of genes related to EMT that includesVimentin, Twist1 and S100A4 levels were also significantly decreased bythe combination treatment (FIG. 22D). This indicates that inhibitoryeffects of combination treatment of cisplatin with endotrophinneutralization mediate a suppression of EMT, lack of which results inloss of crucial traits for metastatic growth.

Discussion

The cellular responses were tested to endotrophin onchemo-responsiveness in mammary tumors treated with cisplatin. It wasdemonstrated that a robust response of cancer cells to cisplatin ishighly dependent on the presence of the endotrophin-driven EMT process.Endotrophin overexpression, leading to enhanced EMT, causes cisplatinresistance. The data presented here suggests that determiningendotrophin levels in association with the EMT status is critical forpredicting cisplatin response. Higher levels of endotrophin occur inadvanced metastatic breast cancers (Iyengar et al, 2005) and contributeto the poor chemo-response. It also suggests that this subset of tumorsis likely to undergo EMT, which plays a major role in tumor progression,metastasis and multi-drug resistance in various epithelial cancer cells(Haslehurst et al, 2012; Latifi et al, 2011; Rosano et al, 2011).Furthermore, it is proposed that obesity is one of the major riskfactors to provide an endotrophin-enriched tumor microenvironment,because it is mainly secreted from adipose tissue and elevated indysfunctional adipose tissue. Therefore, the endotrophin-mediated EMTdescribed here may also be predictive of a poor chemotherapeuticresponse in other types of cancers.

It has been appreciated that there is an enormous degree of ECMremodeling going on in response to chemotherapy, and this in turn has animpact on drug penetration, which critically affects chemo-sensitivity.In addition, increased tissue stiffness seems to confer survival signalsto cancer cells through enhanced anchoring of cancers to ECMs. Beyondthese purely mechanical roles of ECM remodeling, here endotrophin wasfound to act as a signaling molecule leading to an enhanced EMT process,resulting in cisplatin resistance.

The beneficial effects of the combination of TZDs with platinum-basedchemotherapy are appreciated. Based on the data herein, TZD monotherapyfails to have an impact on tumor progression in PyMT mice, and in factfurther enhances growth. This is consistent with clinical reports thatfailed to see an impact on the malignancies of epithelial cancer cells(Burstein et al, 2003; Kulke et al, 2002; Smith et al, 2004). However,TZDs in combination with cisplatin is highly beneficial. How do TZDsenhance cisplatin effectiveness? Here, it is shown that the beneficialeffects of TZDs on cisplatin therapies are due to marked reduction ofthe endotrophin levels. This attenuates the downstream consequences ofendotrophin signaling, including a suppression of EMT, fibrosis andangiogenesis, thereby leading to an increase of chemo-sensitivity (FIG.22E). Therefore, a treatment criterion for a TZD/cisplatin combinationtherapy would be high levels of endotrophin in association with EMT, dueto the fact that the beneficial effects of TZDs are acquired through adirect suppression of endotrophin-induced EMT. Along those lines, it isshown that neutralizing endotrophin activity through the use ofneutralizing monoclonal antibodies during cisplatin treatmenteffectively inhibits the tumor growth and metastasis.

In summary, a rodent model was employed for a chemotherapeutic tumorresponse, and demonstrated that the endotrophin-mediated induction ofthe EMT results in chemo-resistance. Furthermore, the beneficial effectsof TZDs on cisplatin-based therapies are shown to be mediated throughthe suppression of this pathway. These results provide a directexplanation for previous correlations reported in the context of poorresponses to platinum-based chemotherapy in tumors expressing highlevels of COLE. This also suggests that endotrophin levels as apromising predictive marker to decide if a TZD combination should beinitiated along with a platinum-based therapeutic approach.

Materials and Methods

Animal Experiments.

All animal experiments were approved by the Institutional Animal Careand Research Advisory Committee at the University of Texas SouthwesternMedical Center. MMTV-PyMT mice (Guy et al, 1992) were used as a mousemammary tumor model. MMTV-Endotrophin transgenic mice and MMTV-FP635(Infrared fluorescent protein FP635) transgenic mice were generated aspreviously described in the study (Park & Scherer, 2012a). Allexperiments were conducted using littermate-controlled female mice. Allanimals used in this study are in a pure FVB background.

Reagents.

Cisplatin (sigma, 479306) was diluted to 1 mg/ml in PBS and wassonicated briefly before injection. The peroxisomeproliferator-activated receptor gamma (PPARγ) agonist rosiglitazone(Avandia, GlaxoSmithKline) was given by diet inclusion at a dose of 20mg/kg/day BW. Anti-mouse endotrophin monoclonal antibodies (10B6, 100μg/mouse) were administered by intraperitoneal injection.

Histological Analysis.

Formalin-fixed paraffin-embedded tissue sections were used forimmunostaining. Deparaffinized tissue slides were stained with rabbitanti-mouse endotrophin, metallothionein (Abcam, Ab12228), E-cadherin(Cell signaling, 24E10), Vimentin (Cell signaling, D21H3) andcytokeratin (Cell Signaling, #4545). For immunofluorescence,fluorescence labeled secondary antibodies were used and counterstainedwith DAPI. Images were acquired using the Leica confocal microscope andanalyzed with ImageJ software. For immunohistochemistry, the reactionwas visualized by the DAB Chromogen-A system (Dako Cytomation) andcounterstained with hematoxylin. Images were acquired using the NikonCool Scope. TUNEL assay was according to the manufacturer's protocol(Trevigen, Inc). To assess functional blood vessels formation in tumortissues, mice were injected with biotinylated tomato-lectin (100 μg,i.v) (Vector laboratories, CA) and perfused lectin was visualized by aCy3-labeled streptoavidin. H&E staining and Masson's Trichrome Cstaining were performed by Dr. John Shelton at the University of TexasSouthwestern Medical Center. Histological analysis was performed withpathologists in the UTSW pathology core facility.

Quantitative RT-PCR.

Total RNA was isolated following tissue homogenization in Trizol(Invitrogen, Carlsbad, Calif.) using a TissueLyser (Qiagen, Valencia,Calif.) and isolated using the RNeasy kit (Qiagen). Total RNA (1 μg) wasreverse transcribed with SuperScript III reverse transcriptase(Invitrogen). Quantitative real-time PCR (qRT-PCR) was performed in theRoche Lightcycler 480. For all qRT-PCR experiments, the results werecalculated using the ΔΔCt method using 36B4 to normalize. Primers forCOL1A1, COL6A1, COL6A2, and COL6A3 were followed in previous report(Khan et al, 2009). Other primer sequences used in this study are listedin Table 1 below.

TABLE 1 Gene Sense Antisense Snail CCCTTCAGGCCACCTTCTTT GTCCAGTAACCACCC(Snai1) GAGGT TGCTG (SEQ ID NO: 19) (SEQ ID NO: 20) SlugCTGTATGGACATCGTCGGCA ACTTACACGCCCCAA (Snai2) G GGATG (SEQ ID NO: 21)(SEQ ID NO: 22) Twist1 CGGCCAGGTACATCGACTTC TGCAGCTTGCCATCTT(SEQ ID NO: 23) GGAG (SEQ ID NO: 24) Twist2 TCAGCAAGATCCAGACGCTCTGAGATGTGCAGGT C GGGTC (SEQ ID NO: 25) (SEQ ID NO: 26) E-CadherinCGATTACGAGGGCAGTGGT AGTCCCCTAGTCGTCC (Cdh1) T TCAC (SEQ ID NO: 27)(SEQ ID NO: 28) N-Cadherin GGCAATCCCACTTATGGCCT TCCGTGACAGTTAGG (Cdh2)TTGGC (SEQ ID NO: 29) (SEQ ID NO: 30) Vimentin GCCAGCAGTATGAAAGCGTACCTGTCTCCGGTACT G CGTT (SEQ ID NO: 31) (SEQ ID NO: 32) S100A4TTGTGTCCACCTTCCACAAA TGTTGCTGTCCAAGTT (SEQ ID NO: 33) GCTC(SEQ ID NO: 34) COL6A3-N ACGCCCATCACCACTCTAAC CTAAACTGCACGACC(SEQ ID NO: 35) CCAAT (SEQ ID NO: 36) 36B4 GGCATGCGGCCCGTCTCTCCTTCCCTGGGCATCAC GGCG (SEQ ID NO: 38) (SEQ ID NO: 37)

Primary Culture of Mammary Cancer Cells and Implantation.

Mammary epithelial cancer cells were isolated as described in previousreport (Park et al, 2010). 1 day after cell culture, same amount ofcancer cells were counted and implanted into inguinal fat-pad of 8- to10-week-old indicated recipient mice by intraductal injection. Tumorgrowth was determined from 10 days after implantation and twice a weekover the course of tumor progression.

Analysis of Tumor Progression.

Tumor onset was monitored twice weekly by palpation. Tumor sizes weremeasured with a digital caliper twice weekly and the volumes werecalculated as (length×width²)/2. Inguinal tumors were weighted todetermine tumor burden. Animals were sacrificed when the tumor burdenvisibly affected the host or when the tumors reached the IACUCpredetermined limit of 20 mm along one axis.

Tumor Imaging.

Infrared fluorescence expressing MMTV-PyMT mice (FP635/PyMT) were imagedby IVIS scanner (Caliper lifesciences) and signal intensity was analyzedwith Living image v. 3.2 (Caliper lifesciences).

Statistical Analyses.

All data represent mean±SD. Data were analyzed by 2-way ANOVA followedby Newman-Keuls multiple comparison test or by Student's t-test andMann-Whitney t-test, as appropriate with GraphPad Prism v. 5 software.P-value less than 0.05 was considered as statistical significance.

Example 3: Antibodies

As shown in FIGS. 28A-28C, an anti-mouse endotrophin monoclonal antibody(10B6 mAb) was prepared using standard hybridoma technology. FIG. 28Aillustrates the purified 10B6 antibody produced by a rat hybridoma cellline expressing an anti-mouse endotrophin monoclonal antibody. FIG. 28Billustrates the transient expression of cloned 10B6 antibody as arat/human chimera in HEK293 cells. FIG. 28C shows the 10B6 Kappa chainsequence (coding DNA (SEQ ID NO: 1) and amino acid (SEQ ID NO: 2)sequences) and alignment with IGKV22S7*01. Based on the alignment, the 3CDR's of the 10B6 Kappa chain are: QNINKY (CDR1; SEQ ID NO: 7), NTN(CDR2) and LQHSSLYT (CDR3; SEQ ID NO: 8). FIG. 28D shows the 10B6 heavychain sequence (coding DNA (SEQ ID NO: 3) and amino acid (SEQ ID NO: 4)sequences) and alignment with IGHV1S12*01. Based on the alignment, the 3CDR's of the 10B6 heavy chain are: GYTFTSYE (CDR1; SEQ ID NO: 9),IYPESGST (CDR2; SEQ ID NO: 10) and TRGLRVLGYVMDV (CDR3; SEQ ID NO: 11).

An anti-human endotrophin monoclonal antibody is also being preparedusing standard hybridoma technology. Such an antibody can be furthermodified into, e.g., a chimeric antibody or a humanized antibody. Theantibody can recognize the human endotrophin target, and providetherapeutic benefits in human cancer patients, such as increasingsensitivity to platinum-based chemotherapy in tumors (or overcomingplatinum-resistance in chemotherapy, alone or together with TZD), andreducing angiogenesis and/or fibrosis in tumor progression. Further,such an antibody can be used as a predictive marker to decide if TZDcombination should be initiated along with a platinum-based therapeuticapproach.

Example 4: Metabolic Effects

A follow up study on the carboxy-terminal endotrophin cleavage productof Col6α3 revealed that abundant secretion of endotrophin from 3T3-L1preadipocytes and fully differentiated adipocytes. Furthermore, it isdemonstrated that endotrophin is up-regulated in the obese state. As anadipocyte-derived and an obesity-associated factor, the direct action ofendotrophin on adipose tissue dysfunction is also important, even in theabsence of a tumor. The local effects of adipocyte derived endotrophinwere examined, and consequently also its impact on systemic metabolicdysregulation. Endotrophin induced by obesity may be associated withadipose tissue fibrosis, macrophage chemotaxis, inflammation and insulinresistance. This is indeed the case as confirmed by the followingexperiments.

Over-Expression of Endotrophin in Adipose Tissue Increases Body WeightGain, Impairs Insulin Sensitivity and Causes Abnormal AdipokineSecretion in HFD-Challenged Mice.

To investigate the metabolic consequences of endotrophin overexpressionin adipose tissue, transgenic mice and wild type littermate controlswere challenged with HFD for 8 weeks. During the 8-week HFD exposure,endotrophin expressing mice gained more weight and exhibited reducedglucose tolerance and insulin sensitivity. Circulating adiponectinlevels dramatically decreased while leptin levels in serum significantlyincreased in endotrophin transgenic mice. Collectively, over-expressionof endotrophin specifically in adipose tissue impairs proper function ofadipocytes and hence causes systemic metabolic dysfunction. To determinewhether the endotrophin overexpression in AT also affects lipidmetabolism, plasma triglycerides and non-esterified free fatty acid(NEFA) levels were measured. Both triglycerides and NEFA levels weresignificantly higher in endotrophin transgenic mice. Both triglycerideand cholesterol levels in the liver of endotrophin transgenic mice weredramatically increased. Liver histology also shows a clear-cut increaseof lipid droplet number and size, indicating a severe liver steatosis inthe transgenic animals. Over-expression of endotrophin thereforedisplays to a large extent abnormal in lipid metabolism and hence causesthe steatosis in other tissues.

Neutralization of Endotrophin Activities in Diet Induced Obese MiceImproves Whole Body Insulin Sensitivity.

To evaluate the therapeutic potential of endotrophin neutralizingmonoclonal antibodies on metabolic perspectives, diet induced obese(DIO) mice were chronically treated with either IgG control or 10B6 (ratanti-mouse endotrophin monoclonal antibody) via intraperitonealinjection at 2 weeks after high-fat diet (HFD) challenge and maintainedthem on a HFD with antibody treatment for another 4 weeks. Body weightwas comparable between two groups (IgG and 10B6) over the antibodytreatment (FIG. 24A). To examine the effects of endotrophinneutralization on insulin sensitivity, hyperinsulinemic-euglycemicclamps were performed on DIO mice given either IgG or 10B6. Strikingly,10B6 treated DIO mice improved whole-body insulin sensitivity, asdetermined by the amount of glucose required to maintain euglycemia(FIG. 24B) and by the amount of suppression of hepatic glucose efflux(FIGS. 24C and D).

The Levels of Endotrophin in Adipose Tissues are Negatively Correlatedwith Insulin Sensitivity in Obese Human Patients.

Endotrophin immunostaining for human mesenteric adipose tissues showsthat endotrophin is upregulated mostly as a function of insulinsensitivity, not so much as a mere consequence of obesity (FIG. 25).

Neutralization of Endotrophin Activities in Diet Induced Obese MiceImproves Serum and Hepatic Lipid Parameters.

To evaluate the therapeutic potential of endotrophin neutralizingmonoclonal antibodies on lipid parameters, a separate cohort of dietinduced obese (DIO) mice were chronically treated with either IgGcontrol or 10B6 (rat anti-mouse endotrophin monoclonal antibody) viaintraperitoneal injection at 2 weeks after high-fat diet (HFD) challengeand maintained them on a HFD with antibody treatment for another 4weeks, similar to the experiment described in FIG. 24. Body weight wascomparable between two groups (IgG and 10B6) over the antibody treatment(FIG. 26A). DIO mice given 10B6 display dramatically improved the levelsof serum triglyceride (FIG. 26B) and free fatty acids (FIG. 26C) whereasthe serum cholesterol levels were less affected by endotrophinneutralization (FIG. 26D). Additionally, the levels of hepatictriglycerides (FIG. 26E) were also improved whereas the cholesterollevels were comparable between two groups (FIG. 26F). Histologicalanalysis with H&E stains indicates that hepatic lipid accumulation inDIO mice was significantly reduced in the presence of 10B6 compared toIgG control (FIG. 26G).

Collectively, these data on 10B6 treated DIO mice suggest thatendotrophin neutralization improves metabolic profiles, such as thelevels of circulating triglycerides and free fatty acids, reduceshepatic triglyceride levels and also improves systemic insulinsensitivity by reducing hepatic glucose output. Thus, anti-endotrophinagents (e.g., antibodies or fragments thereof as discussed above) can beused to treat various metabolic disorders-related diseases (e.g.,diabetes and obesity).

EQUIVALENTS

The present invention provides among other things novel antibodies andmethods for use in cancer therapeutics. While specific embodiments ofthe subject invention have been discussed, the above specification isillustrative and not restrictive. Many variations of the invention willbecome apparent to those skilled in the art upon review of thisspecification. The full scope of the invention should be determined byreference to the claims, along with their full scope of equivalents, andthe specification, along with such variations.

INCORPORATION BY REFERENCE

All publications, patents and sequence database entries mentioned hereinare hereby incorporated by reference in their entirety as if eachindividual publication or patent was specifically and individuallyindicated to be incorporated by reference.

REFERENCES

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1.-22. (canceled)
 23. A polynucleotide comprising a nucleotide sequencethat encodes: (i) a V_(L) domain comprising V_(L) CDRs having an aminoacid sequence of QNINKY (CDR1; SEQ ID NO: 7), NTN (CDR2) and LQHSSLYT(CDR3; SEQ ID NO: 8); and/or (ii) a V_(H) domain comprising V_(H) CDRshaving an amino acid sequence of GYTFTSYE (CDR1; SEQ ID NO: 9), IYPESGST(CDR2; SEQ ID NO: 10) and TRGLRVLGYVMDV (CDR3; SEQ ID NO: 11).
 24. Thepolynucleotide of claim 23, comprising a nucleotide sequence thatencodes the V_(L) domain comprising V_(L) CDRs having an amino acidsequence of QNINKY (CDR1; SEQ ID NO: 7), NTN (CDR2) and LQHSSLYT (CDR3;SEQ ID NO: 8).
 25. The polynucleotide of claim 23, comprising anucleotide sequence that encodes the V_(L) domain of SEQ ID NO:
 2. 26.The polynucleotide of claim 23, comprising a nucleotide sequence of SEQID NO:
 1. 27. The polynucleotide of claim 23, comprising a nucleotidesequence that encodes the V_(H) domain comprising V_(H) CDRs having anamino acid sequence of GYTFTSYE (CDR1; SEQ ID NO: 9), IYPESGST (CDR2;SEQ ID NO: 10) and TRGLRVLGYVMDV (CDR3; SEQ ID NO: 11).
 28. Thepolynucleotide of claim 23, comprising a nucleotide sequence thatencodes the V_(H) domain of SEQ ID NO:
 4. 29. The polynucleotide ofclaim 23, comprising a nucleotide sequence of SEQ ID NO:
 3. 30. Thepolynucleotide of claim 23, comprising a nucleotide sequence thatencodes: (i) a V_(L) domain comprising V_(L) CDRs having an amino acidsequence of QNINKY (CDR1; SEQ ID NO: 7), NTN (CDR2) and LQHSSLYT (CDR3;SEQ ID NO: 8); and (ii) a V_(H) domain comprising V_(H) CDRs having anamino acid sequence of GYTFTSYE (CDR1; SEQ ID NO: 9), IYPESGST (CDR2;SEQ ID NO: 10) and TRGLRVLGYVMDV (CDR3; SEQ ID NO: 11).
 31. Thepolynucleotide of claim 23, wherein the polynucleotide encodes a Fab′, aF(ab′)2, a F(ab′)3, a monovalent scFv, a bivalent scFv, or a singledomain antibody.
 32. The polynucleotide of claim 23, wherein thepolynucleotide encodes a humanized antibody.
 33. The polynucleotide ofclaim 23, wherein the polynucleotide encodes a IgG antibody.
 34. Acomposition comprising a first polynucleotide encoding a V_(L) domaincomprising V_(L) CDRs having an amino acid sequence of QNINKY (CDR1; SEQID NO: 7), NTN (CDR2) and LQHSSLYT (CDR3; SEQ ID NO: 8); and a secondpolynucleotide encoding a V_(H) domain comprising V_(H) CDRs having anamino acid sequence of GYTFTSYE (CDR1; SEQ ID NO: 9), IYPESGST (CDR2;SEQ ID NO: 10) and TRGLRVLGYVMDV (CDR3; SEQ ID NO: 11).
 35. A cellcomprising a polynucleotide according to claim
 1. 36. A method forproducing an antibody comprising: (a) expressing a polynucleotidemolecule(s) of claim 30 encoding the V_(L) and V_(H) domains of anantibody in a cell; and (b) purifying the antibody from the cell.
 37. Amethod for producing an antibody comprising: (a) expressing apolynucleotide molecule(s) of a composition of claim 34 in a cell; and(b) purifying the antibody from the cell.